Ultralong complementarity determining regions and uses thereof

ABSTRACT

Disclosed herein are immunoglobulin constructs comprising at least one immunoglobulin domain or fragment thereof; and a therapeutic polypeptide or derivative or variant thereof attached to or inserted into said immunoglobulin domain. Also provided are immunoglobulin constructs comprising a mammalian immunoglobulin heavy chain comprising at least a portion of a knob domain in the complementarity-determining region 3 (CDR3H) or fragment thereof; and a therapeutic polypeptide attached to or inserted into said knob domain of the CDR3H. Also provided are immunoglobulin constructs comprising a mammalian immunoglobulin heavy chain comprising at least a portion of a stalk domain in the complementarity-determining region 3 (CDR3H) or fragment thereof; and a therapeutic polypeptide attached to or inserted into said stalk domain of the CDR3H. Also described herein are methods and compositions comprising the immunoglobulin constructs described herein for treatment and prevention of a disease or condition in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/584,680 filed Jan. 9, 2012, and U.S. Provisional Application No.61/671,629, filed Jul. 13, 2012, both of which are incorporated byreference herein in their entirety.

FIELD

Described herein are immunoglobulin constructs comprising at least aportion of an ultralong CDR3, methods of making such constructs,pharmaceutical compositions and medicaments comprising such constructs,and methods of using such constructs and compositions to prevent,inhibit, and/or treat a disease or condition in a subject.

BACKGROUND

Antibodies are natural proteins that the vertebrate immune system formsin response to foreign substances (antigens), primarily for defenseagainst infection. For over a century, antibodies have been induced inanimals under artificial conditions and harvested for use in therapy ordiagnosis of disease conditions, or for biological research. Eachindividual antibody producing cell produces a single type of antibodywith a chemically defined composition, however, antibodies obtaineddirectly from animal serum in response to antigen inoculation actuallycomprise an ensemble of non-identical molecules (e.g., polyclonalantibodies) made from an ensemble of individual antibody producingcells.

Some bovine antibodies have unusually long VH CDR3 sequences compared toother vertebrates. For example, about 10% of IgM contains “ultralong”CDR3 sequences, which can be up to 61 amino acids long. These unusualCDR3s often have multiple cysteines. Functional VH genes form through aprocess called V(D)J recombination, wherein the D-region encodes asignificant proportion of CDR3. A unique D-region encoding an ultralongsequence has been identified in cattle. Ultralong CDR3s are partiallyencoded in the cattle genome, and provide a unique characteristic oftheir antibody repertoire in comparison to humans. Kaushik et al. (U.S.Pat. Nos. 6,740,747 and 7,196,185) disclose several bovine germlineD-gene sequences unique to cattle stated to be useful as probes and abovine VDJ cassette stated to be useful as a vaccine vector.

SUMMARY

The present disclosure provides antibodies that comprise an utralongCDR3 including, libraries that comprise an ultralong CDR3, and usesthereof.

The present disclosure also provides a library of antibodies or bindingfragments thereof, wherein the antibodies or binding fragments thereofcomprise an ultralong CDR3.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 is 35 amino acids in length or longer,40 amino acids in length or longer, 45 amino acids in length or longer,50 amino acids in length or longer, 55 amino acids in length or longer,or 60 amino acids in length or longer.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 is 35 amino acids in length or longer.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises 3 or more cysteine residues, 4or more cysteine residues, 5 or more cysteine residues, 6 or morecysteine residues, 7 or more cysteine residues, 8 or more cysteineresidues, 9 or more cysteine residues, 10 or more cysteine residues, 11or more cysteine residues, or 12 or more cysteine residues.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises 3 or more cysteine residues.

In some embodiments of each or any of the above or below mentionedembodiments, the antibodies or binding fragments thereof comprise acysteine motif.

In some embodiments of each or any of the above or below mentionedembodiments, the cysteine motif is selected from the group consisting ofSEQ ID NOS: 45-156. In some embodiments of each or any of the above orbelow mentioned embodiments, the cysteine motif is selected from thegroup consisting of SEQ ID NOS: 45-99. In some embodiments of each orany of the above or below mentioned embodiments, the cysteine motif isselected from the group consisting of SEQ ID NOS: 45-135. In someembodiments of each or any of the above or below mentioned embodiments,the cysteine motif is selected from the group consisting of SEQ ID NOS:100-135. In some embodiments of each or any of the above or belowmentioned embodiments, the cysteine motif is selected from the groupconsisting of SEQ ID NOS: 136-156.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a non-human DH or a derivativethereof.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a JH sequence or a derivativethereof.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises: a non-human VH sequence or aderivative thereof, a non-human DH sequence or a derivative thereofand/or a JH sequence or derivative thereof.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises an additional amino acidsequence comprising two to six amino acid residues or more positionedbetween the VH sequence and the DH sequence.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a non-bovine sequence.

In some embodiments of each or any of the above or below mentionedembodiments, the non-bovine sequence is a non-antibody or a humansequence.

In some embodiments of each or any of the above or below mentionedembodiments, the non-bovine sequence replaces at least a portion of theultralong CDR3.

In some embodiments of each or any of the above or below mentionedembodiments, the non-bovine sequence is a hormone, lymphokine,interleukin, chemokine, cytokine, toxin, or combination thereof.

In some embodiments of each or any of the above or below mentionedembodiments, the non-bovine sequence is a cytokine.

In some embodiments of each or any of the above or below mentionedembodiments, the cytokine is granulocyte colony-stimulating factor(G-CSF).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises an additional sequence that isa linker.

In some embodiments of each or any of the above or below mentionedembodiments, the linker is linked to a C-terminus, a N-terminus, or bothC-terminus and N-terminus of the non-antibody sequence.

In some embodiments of each or any of the above or below mentionedembodiments, the linker is (GGGGS)_(n)(SEQ ID NO: 339), where n is aninteger between 0 and 5. Alternatively, or additionally, the linker is(GSG)_(n) (SEQ ID NO: 342), GGGSGGGGS (SEQ ID NO: 337) or GGGGSGGGS (SEQID NO: 338)

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a X¹X²X³X⁴X⁵X_(n) motif,wherein X₁ is threonine (T), glycine (G), alanine (A), serine (S), orvaline (V), wherein X₂ is serine (S), threonine (T), proline (P),isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein X₃is valine (V), alanine (A), threonine (T), or aspartic acid (D), whereinX₄ is histidine (H), threonine (T), arginine (R), tyrosine (Y),phenylalanine (F), or leucine (L), wherein X₅ is glutamine (Q), andwherein n is 27-54.

In some embodiments of each or any of the above or below mentionedembodiments, the X¹X²X³X⁴X⁵ motif is TTVHQ (SEQ ID NO: 159) or TSVHQ(SEQ ID NO: 160).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 further comprises a (X^(a)X^(b))_(z)motif, wherein X^(a) is any amino acid residue, X^(b) is an aromaticamino acid selected from the group consisting of: tyrosine (Y),phenylalanine (F), tryptophan (W), and histidine (H), and wherein z is1-4.

In some embodiments of each or any of the above or below mentionedembodiments, the (X^(a)X^(b))_(z) motif is YXYXYX.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a X¹X² _(X) ³ _(X) ⁴ _(X)⁵X_(n)(X^(a)X^(b))_(z) motif, wherein X¹ is threonine (T), glycine (G),alanine (A), serine (S), or valine (V), wherein X² is serine (S),threonine (T), proline (P), isoleucine (I), alanine (A), valine (V), orasparagine (N), wherein X³ is valine (V), alanine (A), threonine (T), oraspartic acid (D), wherein X⁴ is histidine (H), threonine (T), arginine(R), tyrosine (Y), phenylalanine (F), or leucine (L), and wherein X⁵ isglutamine (Q), wherein X^(a) is any amino acid residue, X^(b) is anaromatic amino acid selected from the group consisting of: tyrosine (Y),phenylalanine (F), tryptophan (W), and histidine (H), wherein n is27-54, and wherein z is 1-4.

In some embodiments of each or any of the above or below mentionedembodiments, the X¹X²X³X⁴X⁵ motif is TTVHQ (SEQ ID NO: 159) or TSVHQ(SEQ ID NO: 160), and wherein the (X^(a)X^(b))_(z) motif is YXYXYX.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises TSVHQETKKYQ (SEQ ID NO. 157).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises VHQETKKYQ (SEQ ID NO: 158).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CTTVHQX_(n) (SEQ ID NO. 223).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CTSVHQX_(n) (SEQ ID NO. 223),wherein n is 1-8.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises TTVHQ (SEQ ID NO. 159).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises TSVHQ (SEQ ID NO. 160).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises VHQ.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises KKQ.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises VYQ.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CX¹ X² X³ X⁴Q (SEQ ID NO:228), wherein X¹ is T, S, A, or G, wherein X² is T, S, A, P, or I,wherein X³ is V or K, and wherein X⁴ is H, K, or Y.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CX¹ X²VHQ (SEQ ID NO: 230),wherein X¹ is T, S, A, or G, and wherein X² is T, S, A, P, or I.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CX¹ X²VX³Q (SEQ ID NO: 232),wherein X¹ is T, S, A, or G, wherein X² is T, S, A, P, or I, and whereinX³ is H, Y, or K.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CX¹X²KKQ (SEQ ID NO: 234),wherein X¹ is T, S, A, or G, and wherein X² is T, S, A, P, or I.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises YTYNYEW (SEQ ID NO: 235).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises YX¹YX² (SEQ ID NO: 296),wherein X² is E or D.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises YX¹YX² Y (SEQ ID NO: 297).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises YEX, wherein X is H, W, N, F,I or Y.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises YDX, wherein X is H, W, N, F,I or Y.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises XYE, wherein X is T, S, N orI.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises XYD, wherein X is T, S, N orI.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises Y(E/D)X¹X_(n)W (SEQ ID NOS:304-305), wherein X¹ is H, W, N, F, I or Y, and wherein n is 1-4.

In some embodiments of each or any of the above or below mentionedembodiments, the antibodies or binding fragments thereof are chimeric,human engineered, or humanized.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 is a ruminant CDR3.

In some embodiments of each or any of the above or below mentionedembodiments, the ruminant is a cow.

The present disclosure also provides a library of polynucleotidesencoding for antibodies or binding fragments thereof, wherein theencoded antibodies or binding fragments thereof comprise an ultralongCDR3.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 is 35 amino acids in length or longer,40 amino acids in length or longer, 45 amino acids in length or longer,50 amino acids in length or longer, 55 amino acids in length or longer,or 60 amino acids in length or longer.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 is 35 amino acids in length or longer.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises 3 or more cysteine residues, 4or more cysteine residues, 5 or more cysteine residues, 6 or morecysteine residues, 7 or more cysteine residues, 8 or more cysteineresidues, 9 or more cysteine residues, 10 or more cysteine residues, 11or more cysteine residues, or 12 or more cysteine residues.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises 3 or more cysteine residues.

In some embodiments of each or any of the above or below mentionedembodiments, the antibodies or binding fragments thereof comprise acysteine motif.

In some embodiments of each or any of the above or below mentionedembodiments, the cysteine motif is selected from the group consisting ofSEQ ID NOS: 45-156. In some embodiments of each or any of the above orbelow mentioned embodiments, the cysteine motif is selected from thegroup consisting of SEQ ID NOS: 45-99. In some embodiments of each orany of the above or below mentioned embodiments, the cysteine motif isselected from the group consisting of SEQ ID NOS: 45-135. In someembodiments of each or any of the above or below mentioned embodiments,the cysteine motif is selected from the group consisting of SEQ ID NOS:100-135. In some embodiments of each or any of the above or belowmentioned embodiments, the cysteine motif is selected from the groupconsisting of SEQ ID NOS: 136-156.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a non-human DH or a derivativethereof.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a JH sequence or a derivativethereof.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises: a non-human VH sequence or aderivative thereof, a non-human DH sequence or a derivative thereofand/or a JH sequence or derivative thereof.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises an additional amino acidsequence comprising two to six amino acid residues or more positionedbetween the VH sequence and the DH sequence.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a non-bovine sequence.

In some embodiments of each or any of the above or below mentionedembodiments, the non-bovine sequence is a non-antibody or a humansequence.

In some embodiments of each or any of the above or below mentionedembodiments, the non-bovine sequence replaces at least a portion of theultralong CDR3.

In some embodiments of each or any of the above or below mentionedembodiments, the non-antibody sequence is a hormone, lymphokine,interleukin, chemokine, cytokine, toxin, or combination thereof.

In some embodiments of each or any of the above or below mentionedembodiments, the non-bovine sequence is a cytokine.

In some embodiments of each or any of the above or below mentionedembodiments, the cytokine is granulocyte colony-stimulating factor(G-CSF).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises an additional sequence that isa linker.

In some embodiments of each or any of the above or below mentionedembodiments, the linker is linked to a C-terminus, a N-terminus, or bothC-terminus and N-terminus of the non-antibody sequence.

In some embodiments of each or any of the above or below mentionedembodiments, the linker is (GGGGS)_(n) (SEQ ID NO: 339), where n is aninteger between 0 and 5. Alternatively, the linker is (GSG)_(n) (SEQ IDNO: 342), GGGSGGGGS (SEQ ID NO: 337) or GGGGSGGGS (SEQ ID NO: 338).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a X¹-X²-X³-X⁴-X⁵ motif,wherein X¹ is threonine (T), glycine (G), alanine (A), serine (S), orvaline (V), wherein X² is serine (S), threonine (T), proline (P),isoleucine (I), alanine (A), valine (V), or asparagine (N), wherein X₃is valine (V), alanine (A), threonine (T), or aspartic acid (D), whereinX⁴ is histidine (H), threonine (T), arginine (R), tyrosine (Y),phenylalanine (F), or leucine (L), and wherein X₅ is glutamine (Q).

In some embodiments of each or any of the above or below mentionedembodiments, the X¹X²X³X⁴X⁵ motif is TTVHQ (SEQ ID NO:159) or TSVHQ (SEQID NO: 160).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 further comprises a (X^(a)X^(b))_(z)motif, wherein X^(a) is an aromatic amino acid selected from the groupconsisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), andhistidine (H), wherein X^(b) is any amino acid residue, and wherein z is1-4.

In some embodiments of each or any of the above or below mentionedembodiments, the (X^(a)X^(b))_(z) motif is YXYXYX.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a X¹X²X³X⁴X⁵X_(n)(X⁶X⁷)_(z)motif, wherein X¹ is threonine (T), glycine (G), alanine (A), serine(S), or valine (V), wherein X² is serine (S), threonine (T), proline(P), isoleucine (I), alanine (A), valine (V), or asparagine (N), whereinX³ is valine (V), alanine (A), threonine (T), or aspartic acid (D),wherein X⁴ is histidine (H), threonine (T), arginine (R), tyrosine (Y),phenylalanine (F), or leucine (L), and wherein X⁵ is glutamine (Q),wherein X^(a) is any amino acid residue, X^(b) is an aromatic amino acidselected from the group consisting of: tyrosine (Y), phenylalanine (F),tryptophan (W), and histidine (H), wherein n is 27-54, and wherein z is1-4.

In some embodiments of each or any of the above or below mentionedembodiments, the X¹X²X³X⁴X⁵ motif is TTVHQ (SEQ ID NO: 159) or TSVHQ(SEQ ID NO: 160), and wherein the (X^(a)-X^(b))_(z) motif is YXYXYX.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises TSVHQETKKYQ (SEQ ID NO. 157).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises VHQETKKYQ (SEQ ID NO: 158).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CTTVHQXn (SEQ ID NO. 223).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CTSVHQXn (SEQ ID NO. 224),wherein n is 1-8.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises TTVHQ (SEQ ID NO. 159).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises TSVHQ (SEQ ID NO. 160).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises VHQ.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises KKQ.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises VYQ.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CX¹ X² X³ X⁴Q, wherein X¹ isT, S, A, or G, wherein X² is T, S, A, P, or I, wherein X³ is V or K, andwherein X⁴ is H, K, or Y.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CX¹ X²VHQ, wherein X¹ is T, S,A, or G, and wherein X² is T, S, A, P, or I.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CX¹ X²VX³Q, wherein X¹ is T,S, A, or G, wherein X² is T, S, A, P, or I, and wherein X³ is H, Y, orK.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises CX¹ X²KKQ, wherein X¹ is T, S,A, or G, and wherein X² is T, S, A, P, or I.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises YTYNYEW (SEQ ID NO: 235).

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises YX¹YX², wherein X² is E or D.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises YX¹YX² Y.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises YEX, wherein X is H, W, N, F,I or Y.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises YDX, wherein X is H, W, N, F,I or Y.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises XYE, wherein X is T, S, N orI.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises XYD, wherein X is T, S, N orI.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises Y(E/D)X¹X_(n)W, wherein X¹ isH, W, N, F, I or Y, and wherein n is 1-4.

In some embodiments of each or any of the above or below mentionedembodiments, the antibodies or binding fragments thereof are chimeric,human engineered, or humanized.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 is a ruminant CDR3.

In some embodiments of each or any of the above or below mentionedembodiments, the ruminant is a cow.

In some embodiments of each or any of the above or below mentionedembodiments, the antibodies or binding fragments thereof are present ina spatially addressed format.

In some embodiments of each or any of the above or below mentionedembodiments, the polynucleotides coding for the antibodies or bindingfragments thereof are present in a spatially addressed format.

The present disclosure also provides a library of vectors comprising anyof the library of polynucleotides disclosed herein.

The present disclosure also provides a library of host cells comprisingthe any library of vectors disclosed herein.

In some embodiments of each or any of the above or below mentionedembodiments, the cell is a bacteria, virus, or bacteriophage.

In some embodiments of each or any of the above or below mentionedembodiments, the antibodies or binding fragments thereof are displayedon the cell surface.

In some embodiments of each or any of the above or below mentionedembodiments, the antibodies or binding fragments thereof are secretedfrom the cell.

In some embodiments of each or any of the above or below mentionedembodiments, the antibodies or binding fragments thereof are present ina spatially addressed format.

The present disclosure also provides an antibody or binding fragmentthereof comprising an ultralong CDR3, wherein the ultralong CDR3comprises a non-bovine sequence.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 is 35 amino acids in length or longer,40 amino acids in length or longer, 45 amino acids in length or longer,50 amino acids in length or longer, 55 amino acids in length or longer,or 60 amino acids in length or longer.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 is 35 amino acids in length or longer.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises 3 or more cysteine residues, 4or more cysteine residues, 5 or more cysteine residues, 6 or morecysteine residues, 7 or more cysteine residues, 8 or more cysteineresidues, 9 or more cysteine residues, 10 or more cysteine residues, 11or more cysteine residues, or 12 or more cysteine residues.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises 3 or more cysteine residues.

In some embodiments of each or any of the above or below mentionedembodiments, the antibodies or binding fragments thereof comprise acysteine motif.

In some embodiments of each or any of the above or below mentionedembodiments, the cysteine motif is selected from the group consisting ofSEQ ID NOS: 45-156. In some embodiments of each or any of the above orbelow mentioned embodiments, the cysteine motif is selected from thegroup consisting of SEQ ID NOS: 45-99. In some embodiments of each orany of the above or below mentioned embodiments, the cysteine motif isselected from the group consisting of SEQ ID NOS: 45-135. In someembodiments of each or any of the above or below mentioned embodiments,the cysteine motif is selected from the group consisting of SEQ ID NOS:100-135. In some embodiments of each or any of the above or belowmentioned embodiments, the cysteine motif is selected from the groupconsisting of SEQ ID NOS: 136-156.

In some embodiments of each or any of the above or below mentionedembodiments, the non-bovine sequence is a non-antibody sequence or ahuman sequence.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a non-human DH or a derivativethereof.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises a JH sequence or a derivativethereof.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises: a non-human VH sequence or aderivative thereof, a non-human DH sequence or a derivative thereofand/or a JH sequence or derivative thereof.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 comprises an additional amino acidsequence comprising two to six amino acid residues or more positionedbetween the VH sequence and the DH sequence.

In some embodiments of each or any of the above or below mentionedembodiments, the non-bovine sequence replaces at least a portion of theultralong CDR3.

In some embodiments of each or any of the above or below mentionedembodiments, the non-bovine sequence is a hormone, lymphokine,interleukin, chemokine, cytokine, toxin, or combination thereof.

In some embodiments of each or any of the above or below mentionedembodiments, the non-bovine sequence is a cytokine.

In some embodiments of each or any of the above or below mentionedembodiments, the cytokine is granulocyte colony-stimulating factor(G-CSF).

In some embodiments of each or any of the above or below mentionedembodiments, the antibodies or binding fragments thereof are chimeric,human engineered, or humanized.

In some embodiments of each or any of the above or below mentionedembodiments, the ultralong CDR3 is a ruminant CDR3.

In some embodiments of each or any of the above or below mentionedembodiments, the ruminant is a cow.

The present disclosure also provides a polynucleotide encoding for anyantibody or binding fragment thereof disclosed herein.

The present disclosure also provides a vector comprising anypolynucleotide encoding for the antibody or binding fragment thereofdisclosed herein.

The present disclosure also provides a host cell comprising any vectordisclosed herein.

In some embodiments of each or any of the above or below mentionedembodiments, the cell is a bacteria, virus, or bacteriophage.

In some embodiments of each or any of the above or below mentionedembodiments, the antibody or binding fragment thereof is displayed onthe surface of the cell.

In some embodiments of each or any of the above or below mentionedembodiments, the antibody or binding fragment thereof is secreted fromthe cell.

The present disclosure also provides a method of producing an antibodyor binding fragment thereof comprising an ultralong CDR3 or fragmentthereof comprising culturing a host cell comprising a polynucleotideencoding any of the antibodies disclosed herein under conditions whereinthe polynucleotide sequence is expressed and the antibody or bindingfragment thereof comprising an ultralong CDR3 or fragment thereof isproduced.

In some embodiments of each or any of the above or below mentionedembodiments, the methods further comprise recovering the antibody orbinding fragment thereof comprising an ultralong CDR3 or fragmentthereof from the host cell culture.

The present disclosure also provides a pharmaceutical compositioncomprising any antibody or binding fragment thereof disclosed herein.

The present disclosure also provides a method of treating a mammal inneed thereof comprising administering to the mammal an amount of anyantibody disclosed herein.

In some embodiments of each or any of the above or below mentionedembodiments, the mammal is a human.

In some embodiments is a recombinant antibody or fragment thereof,wherein at least a portion of the recombinant antibody or fragmentthereof is based on or derived from at least a portion of an ultralongCDR3.

In some embodiments is an antibody or fragment thereof comprising atleast a portion of an ultralong CDR3 sequence and at least a portion ofa non-bovine sequence.

In some embodiments is an antibody or fragment thereof comprising (a) afirst antibody sequence, wherein at least a portion of the firstantibody sequence is derived from at least a portion of an ultralongCDR3; (b) a non-antibody sequence; and (c) optionally, a second antibodysequence, wherein at least a portion of the second antibody sequence isderived from at least a portion of an ultralong CDR3.

The antibodies disclosed herein may be a chimeric, human engineered, orhumanized antibody. The antibodies disclosed herein may be a bovineengineered, bovinized, or fully bovine antibody. The antibodiesdisclosed herein may comprise a Fab, a scFv, dsFv, diabody, (dsFv)₂,minibody, flex minibody or bi-specific fragment. The antibodiesdisclosed herein may be an isolated antibody.

The antibodies disclosed herein may further comprise a non-antibodysequence. The non-antibody sequence may be derived from a mammal. Themammal may be a bovine, human, or non-bovine mammal. The antibodiesdisclosed herein may comprise a non-antibody sequence derived from anon-bovine animal. The non-bovine animal may be a scorpion. Thenon-bovine animal may be a lizard. The lizard may be a gila monster. Thenon-antibody sequence may be a derived from a growth factor. The growthfactor may be a GCSF, GMCSF or FGF21. The GCSF may be a bovine GCSF.Alternatively, the GCSF may be a human GCSF. The GMCSF and/or the FGF21may be from a human. The non-antibody sequence may be a derived from acytokine. The cytokine may be a beta-interferon. The non-antibodysequence may be a derived from a hormone. The hormone may be anexendin-4, GLP-1, somatostatin, or erythropoietin. The GLP-1 and/orerythropoietin may be from a human. The non-antibody sequence may be aderived from a toxin. The toxin may be a Moka1, VM-24, ziconotide,chlorotoxin, or protoxin2 (ProTxII). The non-antibody sequence may beIL8, ziconotide, somatostatin, chlorotoxin, SDF1(alpha), or IL21. Thenon-antibody sequence may comprise an amino acid sequence based on orderived from any of SEQ ID NOS: 317-332. The non-antibody sequence mayreplace at least a portion of the ultralong CDR3. The non-antibodysequence may be inserted into the sequence of the ultralong CDR3. Thenon-antibody sequence may be conjugated to at least a portion of theantibody (e.g., ultralong CDR3, variable region, heavy chain, lightchain). The non-antibody sequence may be attached to the ultralong CDR3,linker, cleavage site, non-bovine sequence, non-ultralong CDR3 antibodysequence, or combination thereof. The non-antibody sequence may beadjacent to the ultralong CDR3, linker, cleavage site, non-bovinesequence, non-ultralong CDR3 antibody sequence, or combination thereof.

The antibodies disclosed herein may comprise an ultralong CDR3 may bebased on or derived from a cow ultralong CDR3. At least a portion of theantibodies disclosed herein may be from a mammal. At least a portion ofthe first antibody sequence and/or at least a portion of the secondantibody sequence of the antibodies disclosed herein may be from amammal. The mammal may be a bovine, human or non-bovine mammal.

The antibodies disclosed herein may comprise 3 or more amino acids inlength. The antibodies disclosed herein may comprise a sequence that maybe based on or derived from an ultralong CDR3 disclosed herein. Theantibodies disclosed herein may comprise 1 or more amino acid residuesbased on or derived from a stalk domain of the ultralong CDR3. Theantibodies disclosed herein may comprise 1 or more amino acid residuesbased on or derived from a knob domain of the ultralong CDR3.

At least a portion of the antibodies disclosed herein may be based on orderived from at least a portion of an ultralong CDR3 disclosed herein.The portion of the antibody based on or derived from at least a portionof the ultralong CDR3 may be 20 or fewer amino acids in length. Theportion of the antibody based on or derived from at least a portion ofthe ultralong CDR3 may be 3 or more amino acids in length

The antibodies disclosed herein may comprise 1 or more conserved motifsderived from a stalk domain of the ultralong CDR3. The 1 or moreconserved motifs derived from the stalk domain of the ultralong CDR3 maycomprise any of the stalk domain conserved motifs disclosed herein.

The portion of the ultralong CDR3s disclosed herein may comprise atleast a portion of a stalk domain of the ultralong CDR3, at least aportion of the knob domain of the ultralong CDR3, or a combinationthereof.

The antibodies disclosed herein may comprise a sequence selected fromany one of SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The antibodiesdisclosed herein may comprise a sequence that may be 50% or morehomologous to a sequence selected from any one of SEQ ID NOS: 157-224and 235-295.

A portion of any of the antibodies disclosed herein may be based on orderived from at least a portion of a single ultralong CDR3 sequence. Aportion of the antibodies disclosed herein may be based on or derivedfrom at least a portion of two or more different ultralong CDR3sequences.

In any of the embodiments disclosed herein, the portion of the ultralongCDR3 is based on or derived from a BLV1H12 ultralong CDR3 sequence. Theportion of the ultralong CDR3 may be based on or derived from a sequencethat may be 50% or more homologous to a BLV1H12 ultralong CDR3 sequence.The portion of the ultralong CDR3 may be based on or derived from aBLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralong CDR3 sequence.The portion of the ultralong CDR3 may be based on or derived from asequence that may be 50% or more homologous to a BLV5B8, BLVCV1, BLV5D3,BLV8C11, BF1H1, or F18 ultralong CDR3 sequence.

The antibodies disclosed herein may comprise a first and/or secondantibody sequence that comprises 3 or more amino acids in length. Aportion of the first antibody sequence derived from at least a portionof the ultralong CDR3 and/or the portion of the second antibody sequencederived from at least a portion of the ultralong CDR3 may be 20 or feweramino acids in length. A portion of the first antibody sequence derivedfrom at least a portion of the ultralong CDR3 and/or the portion of thesecond antibody sequence derived from at least a portion of theultralong CDR3 may be 3 or more amino acids in length.

In any of the embodiments disclosed herein, the first and/or secondantibody sequences comprise one or more amino acid residues based on orderived from a stalk domain of the ultralong CDR3. The first and/orsecond antibody sequences may comprise one or more amino acid residuesbased on or derived from a knob domain of the ultralong CDR3. The one ormore amino acid residues derived from the knob domain of the ultralongCDR3 may be a serine and/or cysteine residue. The first and/or secondantibody sequences may comprise one or more conserved motifs derivedfrom a stalk domain of the ultralong CDR3. The one or more conservedmotifs derived from the stalk domain of the ultralong CDR3 may comprisea sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS:333-336.

In any of the embodiments disclosed herein, the portion of the firstantibody sequence derived from at least a portion of the ultralong CDR3and/or the portion of the second antibody sequence derived from at leasta portion of the ultralong CDR3 comprises a sequence selected from anyone of SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The portion of thefirst antibody sequence derived from at least a portion of the ultralongCDR3 and/or the portion of the second antibody sequence derived from atleast a portion of the ultralong CDR3 may comprise a sequence that maybe 50% or more homologous to a sequence selected from any one of SEQ IDNOS: 157-224 and 235-295. The portion of the first antibody sequencederived from at least a portion of the ultralong CDR3 may comprise asequence selected from any one of SEQ ID NOS: 157-234. The portion ofthe first antibody sequence derived from at least a portion of theultralong CDR3 may comprise a sequence that may be 50% or morehomologous to a sequence selected from any one of SEQ ID NOS: 157-224.

In any of the embodiments disclosed herein, the portion of the secondantibody sequence derived from at least a portion of the ultralong CDR3comprises a sequence selected from any one of SEQ ID NOS: 235-307 andSEQ ID NOS: 333-336. The portion of the second antibody sequence derivedfrom at least a portion of the ultralong CDR3 may comprise a sequencethat may be 50% or more homologous to a sequence selected from any oneof SEQ ID NOS: 235-295. The portion of the first antibody sequencederived from at least a portion of the ultralong CDR3 and the portion ofthe second antibody sequence derived from at least a portion of theultralong CDR3 may be derived from the same ultralong CDR3 sequence.

In any of the embodiments disclosed herein, the portion of the firstantibody sequence derived from at least a portion of the ultralong CDR3and the portion of the second antibody sequence derived from at least aportion of the ultralong CDR3 is derived from two or more differentultralong CDR3 sequences. The portions of the ultralong CDR3 of thefirst and/or second antibody sequences may be based on or derived from aBLV1H12 ultralong CDR3 sequence. The portions of the ultralong CDR3 ofthe first and/or second antibody sequences may be based on or derivedfrom a sequence that may be 50% or more homologous to a BLV1H12ultralong CDR3 sequence. The portions of the ultralong CDR3 of the firstand/or second antibody sequences may be based on or derived from aBLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralong CDR3 sequence.The portions of the ultralong CDR3 of the first and/or second antibodysequences may be based on or derived from a sequence that may be 50% ormore homologous to a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18ultralong CDR3 sequence.

In any of the embodiments disclosed herein, the ultralong CDR3 is basedon or derived from an ultralong CDR3 that may be 35 or more amino acidsin length. The ultralong CDR3 may be based on or derived from anultralong CDR3 comprising 3 or more cysteine residues.

In any of the embodiments disclosed herein, the ultralong CDR3 is basedon or derived from an ultralong CDR3 may comprise one or more cysteinemotifs. The one or more cysteine motifs may be selected from the groupconsisting of SEQ ID NOS: 45-156. In some embodiments of each or any ofthe above or below mentioned embodiments, the cysteine motif is selectedfrom the group consisting of SEQ ID NOS: 45-99. In some embodiments ofeach or any of the above or below mentioned embodiments, the cysteinemotif is selected from the group consisting of SEQ ID NOS: 45-135. Insome embodiments of each or any of the above or below mentionedembodiments, the cysteine motif is selected from the group consisting ofSEQ ID NOS: 100-135. In some embodiments of each or any of the above orbelow mentioned embodiments, the cysteine motif is selected from thegroup consisting of SEQ ID NOS: 136-156.

The antibodies disclosed herein may be based on or derived from anultralong CDR3 that may be 35 or more amino acids in length. Theantibodies disclosed herein may be based on or derived from an ultralongCDR3 comprising 3 or more cysteine residues. The antibodies disclosedherein may be based on or derived from an ultralong CDR3 may comprise 1or more cysteine motifs.

The antibodies disclosed herein may comprise an ultralong CDR3 that is35 or more amino acids in length. The antibodies disclosed herein maycomprise an ultralong CDR3 comprising 3 or more cysteine residues. Theantibodies disclosed herein may comprise an ultralong CDR3 comprisingone or more cysteine motifs.

In any of the embodiments disclosed herein, the ultralong CDR3 may be aheavy chain CDR3. The ultralong CDR3 may comprise an amino acid sequencederived from or based on a non-human DH sequence. The ultralong CDR3 maycomprise an amino acid sequence derived from or based on a JH sequence.The ultralong CDR3 may comprise an amino acid sequence derived from orbased on a non-human VH sequence; an amino acid sequence derived from orbased on a non-human DH sequence; and/or an amino acid sequence derivedfrom or based on a JH sequence. The ultralong CDR3 may comprise anadditional amino acid sequence comprising at least about two amino acidresidues positioned between the VH derived amino acid sequence and theDH derived amino acid sequence.

Any of the antibodies disclosed herein may comprise a sequence based onor derived from a sequence selected from SEQ ID NOS: 24-44, the antibodyor binding fragment thereof encoded by a DNA sequence based on orderived from the DNA of SEQ ID NOS: 2-22. Any of the antibodiesdisclosed herein may comprise a sequence based on or derived from asequence selected from SEQ ID NO: 23, the antibody or binding fragmentthereof encoded by a DNA sequence based on or derived from the DNA ofSEQ ID NO: 1.

Any of the ultralong CDR3s disclosed herein may comprise a sequencebased on or derived from a sequence selected from SEQ ID NOS: 24-44. Anyof the antibodies disclosed herein may comprise a sequence based on orderived from a sequence selected from SEQ ID NO: 23. Any of theultralong CDR3s disclosed herein may be encoded by a DNA sequence thatmay be derived from or based on SEQ ID NOS: 2-22. Any of the antibodiesdisclosed herein may comprise a portion encoded by a DNA sequence thatmay be derived from or based on SEQ ID NO: 1.

Any of the antibodies disclosed herein may comprise one or more linkers.Any of the antibodies disclosed herein may comprise a first linkersequence. Any of the antibodies disclosed herein may comprise a secondlinker sequence. The first and second linker sequences may comprise thesame sequence. The first and second linker sequences may comprisedifferent sequences. The first and/or second linker sequences may be thesame length. The first and/or second linker sequences may be differentlengths. The first and/or second linker sequences may be 3 or more aminoacids in length.

The first and/or second linker sequence may attach the non-antibodysequence to the portion based on or derived from the portion of theultralong CDR3. The first and/or second linker sequences may attach thenon-antibody sequence to the first antibody sequence. The first and/orsecond linker sequences may attach the non-antibody sequence to thesecond antibody sequence. The first and/or second linker sequences maybe adjacent to a non-antibody sequence, a portion of an ultralong CDR3sequence, a cleavage site sequence, a non-bovine sequence, an antibodysequence, or a combination thereof.

The first and/or second linker sequences may comprise one or moreglycine residues. The first and/or second linker sequences may comprisetwo or more consecutive glycine residues. The first and/or second linkersequences may comprise one or more serine residues. The first and/orsecond linker sequence may comprise one or more polar amino acidresidues. The one or more polar amino acid residues may be selected fromserine, threonine, asparagine, or glutamine. The polar amino acidresidues may comprise uncharged side chains. The first and/or secondlinker sequences may comprise the sequence (GGGGS)_(n), wherein n=1 to5; the sequence GGGSGGGGS; the sequence GGGGSGGGS; the sequence of(GSG)n, wherein n is greater than or equal to one; or a combinationthereof.

Any of the antibodies disclosed herein may comprise one or more cleavagesites. The one or more cleavage sites may comprise a recognition sitefor a protease. The protease may be a Factor Xa or thrombin. The one ormore cleavage sites may comprise an amino acid sequence of IEGR.

The one or more cleavage site may be between a first antibody sequenceand the non-antibody sequence. The one or more cleavage sites may bebetween a second antibody sequence and the non-antibody sequence. Theone or more cleavage sites may be between the one or more linkers andthe non-antibody sequence. The one or more cleavage sites may be betweena first antibody sequence and the one or more linkers. The one or morecleavage sites may be between a second antibody sequence and the one ormore linkers. The one or more cleavage sites may be adjacent to anon-antibody sequence, a portion of an ultralong CDR3 sequence, a linkersequence, an antibody sequence, or a combination thereof.

In some embodiments is library of antibodies or binding fragmentsthereof, wherein the antibodies or binding fragments thereof maycomprise an ultralong CDR3.

In some embodiments is library of antibodies or binding fragmentsthereof, wherein the antibodies or binding fragments thereof maycomprise any of the antibodies disclosed herein.

In some embodiments is nucleic acid library comprising a plurality ofpolynucleotides comprising sequences coding for antibodies or bindingfragments thereof, wherein the antibodies or binding fragments thereofmay comprise an ultralong CDR3.

In some embodiments is nucleic acid library comprising a plurality ofpolynucleotides comprising sequences coding for antibodies or bindingfragments thereof, wherein the antibodies or binding fragments thereofmay comprise any of the antibodies disclosed herein.

In some embodiments is polynucleotide comprising a nucleic acid sequencethat encodes a variable region, wherein the variable region may comprisean ultralong CDR3.

In some embodiments is vector comprising a polynucleotide, wherein thepolynucleotide comprises a nucleic acid sequence that encodes a variableregion, wherein the variable region may comprise an ultralong CDR3.

In some embodiments is host cell comprising a polynucleotide, whereinthe polynucleotide comprises a nucleic acid sequence that encodes avariable region, wherein the variable region may comprise an ultralongCDR3.

In some embodiments is polynucleotide comprising a nucleic acid sequencethat encodes the antibody or binding fragment thereof of any of theantibodies disclosed herein.

In some embodiments is vector comprising a polynucleotide, wherein thepolynucleotide comprises a nucleic acid sequence that encodes theantibody or binding fragment thereof of any of the antibodies disclosedherein.

In some embodiments is host cell comprising a polynucleotide, whereinthe polynucleotide comprises a nucleic acid sequence that encodes theantibody or binding fragment thereof of any of the antibodies disclosedherein.

In some embodiments is method of producing an antibody or bindingfragment thereof comprising an ultralong CDR3 or fragment thereofcomprising culturing a host cell comprising a polynucleotide, whereinthe polynucleotide may comprise a nucleic acid sequence that encodes theantibody or binding fragment thereof of any of the antibodies disclosedherein under conditions wherein the polynucleotide sequence may beexpressed and the antibody or binding fragment thereof comprising anultralong CDR3 or fragment thereof may be produced. The method maycomprise recovering the antibody or binding fragment thereof comprisingthe ultralong CDR3 or fragment thereof from the host cell culture.

In some embodiments is pharmaceutical composition comprising any of theantibodies disclosed herein. The pharmaceutical compostion may comprisetwo or more antibodies, wherein at least one of the two or moreantibodies comprises at least a portion of an ultralong CDR3.

In some embodiments is pharmaceutical composition comprising (a) anantibody or fragment thereof comprising sequence based on or derivedfrom at least a portion of an ultralong CDR3; and (b) a pharmaceuticallyacceptable excipient.

In some embodiments is method of treating a disease or condition in asubject in need thereof comprising administering to the mammal atherapeutically effective amount of any of the antibodies disclosedherein. In some instances, the antibodies disclosed herein comprise anultralong CDR3 sequence and a non-antibody sequence. In some instances,the non-antibody sequence is selected from the group comprising Moka1,Vm24, human GLP-1, Exendin-4, beta-interferon, human EPO, human FGF21,human GMCSF, human interferon-beta, bovine GCSF, human GCSF, be IL8,ziconotide, somatostatin, chlorotoxin, SDF1(alpha), IL21 and aderivative or variant thereof. The non-antibody sequence may comprise anamino acid sequence based on or derived from any of SEQ ID NOS: 317-332.

The disease or condition may be selected from the group comprisingautoimmune disease, heteroimmune disease or condition, inflammatorydisease, pathogenic infection, thromboembolic disorder, respiratorydisease or condition, metabolic disease, central nervous system (CNS)disorder, bone disease, cancer, blood disorder, obesity, diabetes,osteoporosis, anemia, or pain.

The disease or condition would benefit from the modulation of an ionchannel. The ion channel may be selected from the group comprising apotassium ion channel, sodium ion channel, or acid sensing ion channel.The ion channel may be selected from the group comprising Kv1.3 ionchannel, Nav1.7 ion channel and acid sensing ion channel (ASIC).

The disease or condition would benefit from the modulation of areceptor. The receptor may be selected from the group comprising GLP1R,GCGR, EPO receptor, FGFR, FGF21R, CSFR, GMCSFR, IL8R, IL21R and GCSFR.

The disease or condition may be mastitis.

The subject may be a mammal. The mammal may be a bovine or human.

In some embodiments are crystals based on or derived from the antibodiesdisclosed herein. The crystals may have a space group P2₁2₁2₁. In someinstances, the crystal has the unit cell dimensions of “a” between about40 to 80 angstroms, between 45 to about 75 angstroms, or between about50 to about 75 angstroms; “b” between about 40 to 140 angstroms, betweenabout 50 to about 130 angstroms, between about 55 to about 130angstroms; and “c” between 100 to about 350 angstroms, between 120 toabout 340 angstroms, or between about 125 to about 330 angstroms. Thecrystal may comprise a bovine antibody or portion thereof. The crystalmay comprise a Fab fragment based on or derived from a bovine antibody.The crystal may be an isolated crystal.

In some embodiments, is an isolated crystal comprising a bovine antibodyFab fragment comprising SEQ ID NO: 24 and SEQ ID NO: 23, wherein thecrystal has a space group P2₁2₁2₁ and unit cell dimensions of a=71.4angstroms, b=127.6 angstroms and c=127.9 angstroms.

In some embodiments, is an isolated crystal comprising a bovine antibodyFab fragment comprising SEQ ID NO: 340 and SEQ ID NO: 341, wherein thecrystal has a space group P2₁2₁2₁ and unit cell dimensions of a=54.6angstroms, b=53.7 angstroms and c=330.5 angstroms.

In any or all of the above or below disclosure (e.g., antibodies, uses,or methods) or embodiments utilizing an antibody comprising an ultralongCDR3, any antibody comprising an ultralong CDR3 may be used including,for example, any of the above mentioned antibodies comprising anultralong CDR3.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe disclosure, will be better understood when read in conjunction withthe appended figures. For the purpose of illustrating the disclosure,shown in the figures are embodiments which are presently preferred. Itshould be understood, however, that the disclosure is not limited to theprecise arrangements, examples and instrumentalities shown.

FIG. 1A-C present the identification of a new structural domain inbovine antibodies. FIG. 1A shows a comparison of CDR H3 length amongstmurine, human, and bovine repertoires. An ultralong subset of over 60amino acids is uniquely found in bovine heavy chains. FIG. 1B showssequences of representative CDR H3 from murine (mu), human (hu), orbovine sequences from the literature along with six bovine sequences(B-S1 to B-S4, and B-L1 and B-L2) from our deep sequencing results. Theconserved cysteine of framework 3 and tryptophan of framework 4 thatdefine CDR H3 boundaries in all antibody variable regions are shaded ingrey for reference. The lengths of the CDR H3s are indicated at theright. The murine antibodies include D44.1, an anti-HEL antibody, 93F3,an aldolase, and OKT3, a therapeutic antibody targeting human CD3. Thisantibody is unusual in having a free cysteine in CDR H3. The humanantibodies include Yvo, a cryoglobulin, CR6261, an anti-influenza Ahemaglutinin, and PG9, an anti-HIV antibody which has one of the longesthuman CDR H3s. The bovine antibodies represent the ultralong sequencesin the literature, and short sequences for comparison. BLV5B8 andBLV1H12 (indicated in bold) were used in our structure determinations.Relatively conserved TTVHQ and CPDG motifs are in bold. FIG. 1C showscrystal structures of BLV1H12 (left) and BLV5B8 (middle) Fabs comparedto the 93F3 Fab with a “normal” CDR H3 (right). A superlong, twoβ-stranded stalk protrudes from each bovine V_(H) immunoglobulin domainand terminates in an unusual three disulfide-linked knob domain.

FIG. 2A-C present the structural diversity in ultralong bovineantibodies. FIG. 2A shows a comparison of the structure of the two knobsshowing differences in disulfide patterns. Close up views of the knobsof BLV1H12 (left) and BLV5H8 (right) are shown, in addition to atwo-dimensional representation of the knob and its disulfide pattern.Disulfides are in numbered and circled. The sequences of the knobregions are shown below, with cysteines conserved with the D_(H)2germline gene segment underlined. The disulfide pattern is indicatedabove each sequence. FIG. 2B shows an overlay of the variable regions ofBLV1H12 and BLV5B8 shows structural homology in the variable regionsexcept the upper part of the stalk and knob, which are significantlydivergent. FIG. 2C shows surface and charge density representation ofBLV1H12 (left) and BLV5B8 (right) showing different shapes and charge inthe knob region.

FIG. 3A-C present the genetic basis for ultralong antibody formation.FIG. 3A shows the identification of V_(H)BUL, a germline variable regionused in ultralong antibodies. The leader sequence is in light grey,coding sequence is indicated with the amino acid translation above, theintron is in italics, and the unique TTVHQ extension, which forms aportion of the ascending strand of the stalk is in bold. Therecombination signal sequence heptamer and nonamer are underlined. FIG.3B shows the V_(H)BUL region is found on chromosome 21. Partial cattlemetaphase spread (left) and enlarged chromosome 21 (top right) showingthe location of V_(H)BUL region in BTA21q24 by two-color FISH with BACclones 318H2 and 14-74H6. International nomenclature for BTA21 isdepicted at the bottom. FIG. 3C shows a schematic of the bovineimmunoglobulin loci depicting V_(H)BUL, D_(H)2, and V_(λ)1x, which arepreferentially used in ultralong antibodies. The process of V(D)Jrecombination assembles the gene segments to form functional ultralongheavy and light chain genes. (bottom left) The V-D-J regions mapped ontothe BLV1H12 Fab structure. V_(H)BUL is unique in encoding CDR H1 andCDR5H2 residues that interact with the stalk, as well as a TTVHQ motifthat initiates the ascending β-strand. Similarly, the V_(λ)1x lightchain encodes CDR L1 and CDR L2 residues that interact with the stalk.Arrows indicate areas of potential junctional diversity. Relatively longV-D insertions are indicated in purple. It is unclear whether thissequence results from N-additions, gene conversion, or anothermechanism. (bottom right) A detailed depiction of the interactions ofCDR H1, H2, L1, and L2 with the stalk of BLV1H12, as well as thelocation of the YxYxY motif of the descending strand.

FIG. 4A-C depict deep sequence diversity of bovine ultralong V_(H) CDRH3s. FIG. 4A shows the distribution of the number of cysteines in bovineultralong CDR H3s of IgM and IgG. FIG. 4B shows the length distributionof ultralong CDR H3s. Note that clonal sequences selected during animmune response can bias the proportion at any given length. FIG. 4Cshows representative sequences of ultralong bovine V_(H) CDR H3s. Theterminal portion of the V_(H)BUL region is shown, along with junctionaldiversity at the V-D joint, D_(H)2 and J_(H) (top). The sequences ofBLVH12 and BLV5B8 are shown for comparison, followed by 20 ultralong CDRH3 sequences (bottom). Cysteines conserved with D_(H)2 are underlined.The conserved cysteine and tryptophan that define the CDR H3 boundariesin all antibody variable regions are highlighted in grey for reference.The CPDG motif is underlined in grey and the region of the descendingstrand encoding a YxYxY motif is underlined in grey.

FIG. 5A-C show that cysteine mutations contribute to ultralong CDR H3diversity. FIG. 5A shows that the consensus of ultralong CDR H3 deepsequences aligns with D_(H)2. A consensus sequence for three deepsequencing runs (from two cows) were determined, and aligned with oneanother and with D_(H)2. The consensus aligns well except for some areasof insertions/deletions. Thus, either a single D_(H) gene, or highlyrelated genes, produce the diversity of sequences in ultralong CDR H3antibodies. FIG. 5B shows that the D_(H)2 gene region analysis showingresidues that can readily mutate to cysteine, including SH hotspots. Thenucleotide sequence is above and translated amino-acid sequence below.RGYW hotspots, which are recognized by AID for SH and/or geneconversion, are boxed. Nucleotides at positions 3, 15, 19, 21, 25, 27,31, 33, 39, 43, 45, 49, 51, 57, 60, 64, 73, 75, 79, 81, 84, 88, 90, 93,97, 102, 106, 112, 117, 121, 123, 127, 129, 133, 139, and 145 in (B) canbe altered in a single mutation to a cysteine-encoding codon. FIG. 5Cshows affinity maturation groups show mutation to and from cysteine.Several groups of clonally related sequences were identified andanalyzed for somatic hypermutation. Three groups are shown as examples(labeled 1 to 3 on the left). Sequence differences from cysteine arehighlighted in grey. The number of times each sequence is represented inthe cluster is shown at the right.

FIG. 6A-D show bovine antibodies with ultralong CDR H3s bind antigen.FIG. 6A shows immunization experimental scheme for identifyingantigen-specific, ultralong CDR H3 antibodies. Heavy chain variableregion mRNA was isolated, amplified by RT PCR, and paired with theinvariant light chain to produce a small library of IgG produced inHEK293 cells. FIG. 6B shows ELISA of 132 ultralong CDR H3 antibodiesagainst BVDV (left), and binding activity of the “hits” B8, B9, and H12in a titration assay (right). FIG. 6C shows the sequences of B8, H9, andH12 in comparison to BLV1H12 and the germline D_(H)2 region. Lengths (L)of the CDR H3 are indicated at the right. Cysteines conserved withD_(H)2 are underlined. FIG. 6D shows that H12 binds NS2-3 on cells. Aflag-tagged BVDV NS2-3 protein construct was transfected into HEK293Acells and stained with anti-Flag as a positive control (left), the H12antibody (middle), and B8 (right). Binding assays with untransfectedcells are shown on the bottom.

FIG. 7A-B depict a model for ultralong CDR H3 diversification into novelminifolds. FIG. 7A shows a schematic of the D_(H)2 knob with 4 cysteinesis shown on the left, with SH and/or gene conversion leading to amultitude of new cysteine patterns and new loops on the right. FIG. 7Bshows mechanisms for generating antibody diversity. In humans and mice(left), combinatorial diversity through V(D)J recombination andV_(H)-V_(L) pairing creates a multitude of different binding sites,which are further optimized following antigen exposure by somatichypermutation. In cows (right), combinatorial diversity is severelylimited; however, somatic mutation to and from cysteines can reshape the“knob” region, creating substantial structural diversity in ultralongCDR H3s. These antibodies may be further optimized through SH and maybind unique targets such as pores or channels.

FIG. 8A-J depict schemes showing attachment of bovine G-CSF onto theknob domain of a heavy chain region of bovine BLV1H12 antibody to designan immunoglobulin construct described herein. FIG. 8A shows a ribbondiagram of a heavy chain region and light chain region of bovine BLV1H12antibody. The boxed region highlights the extended region of theultralong CDR3 comprising the stalk and knob domain. FIG. 8B shows aribbon diagram of a growth factor (e.g., bovine G-CSF) inserted into orreplacing a portion of the knob domain of a heavy chain region of abovine BLV1H12 antibody. FIG. 8C shows a ribbon diagram of a toxin(e.g., Moka1, VM-24) inserted into or replacing a portion of the knobdomain of a heavy chain region of a bovine BLV1H12 antibody. FIG. 8Dshows a ribbon diagram of a receptor ligand (e.g., GLP-1, exendin-4)inserted into or replacing a portion of the knob domain of a heavy chainregion of a bovine BLV1H12 antibody. FIG. 8E shows a ribbon diagram of ahormone (e.g., erythropoietin) inserted into or replacing a portion ofthe knob domain of a heavy chain region of a bovine BLV1H12 antibody.FIG. 8F shows a cartoon depicting a heavy chain region and light chainregion of bovine BLV1H12 antibody. FIG. 8G shows a cartoon of a growthfactor (e.g., bovine G-CSF) inserted into or replacing a portion of theknob domain of a heavy chain region of a bovine BLV1H12 antibody. FIG.8H shows a cartoon of a toxin (e.g., Moka1, VM-24) inserted into orreplacing a portion of the knob domain of a heavy chain region of abovine BLV1H12 antibody. FIG. 8I shows a cartoon of a receptor ligand(e.g., GLP-1, exendin-4) inserted into or replacing a portion of theknob domain of a heavy chain region of a bovine BLV1H12 antibody. Theleft panel shows both ends of the receptor ligand inserted into orreplacing a portion of the knob domain; the right panel shows theN-terminus of the receptor ligand released from the knob domain aftertreatment with a protease. FIG. 8J shows a cartoon of a hormone (e.g.,erythropoietin) inserted into or replacing a portion of the knob domainof a heavy chain region of a bovine BLV1H12 antibody. Said attachment ofthe growth factor, toxin, receptor ligand, and hormone can be by meansof a polypeptide linker of sequence GGGGS (Ab-peptide L1) or GGGSGGGGSand GGGGSGGGS (Ab-peptide L2). Another construct (Ab-peptide L0) inwhich attachment is not by means of a linker is not shown in thiscartoon.

FIG. 9A-9E present illustrative mouse NFS-60 cell proliferative activityfor the Ab-bGCSF fusion proteins. FIG. 9A-9B depict proliferativeactivity of bovine G-CSF and human G-CSF respectively. FIG. 9C depictsthe lack of proliferative activity for bovine BLV1H12 antibody in theabsence of G-CSF. FIG. 9D depicts proliferative activity of bovine G-CSFinserted into or replacing a portion of the knob domain in the absenceof a linker (Ab-bGCSF L0). FIG. 9E depicts proliferative activity ofbovine G-CSF inserted into or replacing a portion of the knob domain bymeans of a polypeptide linker of sequence GGGGS (Ab-bGCSF L1).

FIG. 10A-10E present human granulocyte progenitor cell proliferativeactivities of the Ab-GCSF fusion proteins. FIG. 10A-10B depictproliferative activity of bovine G-CSF and human G-CSF respectively.FIG. 10C depicts the lack of proliferative activity for bovine BLV1H12antibody (Ab) in the absence of G-CSF. FIG. 10D depicts proliferativeactivity of bovine G-CSF inserted into or replacing a portion of theknob domain in the absence of a linker (Ab-bGCSF L0). FIG. 10E depictsproliferative activity of bovine G-CSF inserted into or replacing aportion of the knob domain, said attachment by means of a polypeptidelinker of sequence GGGGS (Ab-bGCSF L1).

FIG. 11A-11B depict pharmacokinetics of Ab-bGCSF fusion proteins inmice.

FIG. 12A-12B provide Proliferative activities of Ab-bGCSF fusionproteins on mice neutrophils that are blood stained and counted at the10th day post-injection.

FIG. 13A-13C display expression and purification of Ab-bGCSF fusionproteins in Pichia pastoris. FIG. 13A shows a map of Pichia expressionvector for an immunoglobulin construct provided herein. FIG. 13Bprovides a western blot post-induction of expression of theimmunoglobulin constructs Ab-bGCSF L0 and Ab-bGCSF L1. FIG. 13C providesSDS-PAGE gel of purified Ab-bGCSF L0 and Ab-bGCSF L1 expressed in Pichiaat a yield of ˜70 μg per 100 mL culture.

FIG. 14A-14C show vectors for expression of the immunoglobulinconstructs described herein in free style HEK 293 cells. FIG. 14Aprovides a vector of Ab-bGCSF L0 heavy chain for expression in freestyle HEK 293 cells. FIG. 14B provides a vector of Ab-bGCSF L1 heavychain for expression in free style HEK 293 cells. FIG. 14C provides avector of Ab-bGCSF light chain for expression in free style HEK 293cells.

FIG. 15A shows SDS-PAGE gel of purified antibody fusions. FIG. 15ASDS-PAGE gel of purified Ab-bGCSF L0 and Ab-bGCSF L1 proteins from HEK293 cells. FIG. 15B provides a SDS PAGE of the immunoglobulin constructsAb-Protoxin2 comprising a GGGGS linker attached to both ends ofprotoxin2.

FIG. 16A-16B provide SDS PAGE and activities of the immunoglobulinconstructs Ab-Moka1 L0 (no linker) and Ab-Moka1 L1 (linker). FIG. 16Aprovides a SDS PAGE of the immunoglobulin fusion proteins Ab-Moka1 L0and Ab-Moka1 L1. FIG. 16B provides BLV1H12-Moka1 fusion proteinsinhibitory activities on T cells activation

FIG. 17 provides BLV1H12-Moka1 fusion proteins inhibitory activities onhuman peripheral blood mononuclear cells (PBMCs)

FIG. 18A-18C provide SDS PAGE and activities of the immunoglobulinconstructs Ab-VM24 L1 (GGGGS linker) and Ab-VM24 L2 (GGGSGGGGS andGGGGSGGGS linkers). FIG. 18A provides a SDS PAGE of the immunoglobulinfusion proteins Ab-VM24 L1 and Ab-VM24 L2. FIG. 18B providesBLV1H12-VM24 L1 fusion protein inhibitory activities on T cellsactivation. FIG. 18C provides BLV1H12-VM24 L2 fusion protein inhibitoryactivities on T cells activation.

FIG. 19 provides a SDS PAGE of the immunoglobulin constructs Ab-GLP-1and Ab-Exendin-4.

FIG. 20 provides activity of Ab-GLP-1 and Ab-Ex4 on HEK293 cellsexpressing GLP-1 receptor.

FIG. 21 provides proliferative activity of Ab-hEPO fusion proteins onTF1 cells.

FIG. 22A-C depicts ultralong CDR3 sequences. (Top) Translation from thegermline V_(H)BUL, D_(H)2, and J_(H). The 5 full length ultralong CDRH3s reported in the literature contain between four and eight cysteinesand are not highly homologous to one another; however, some conservationof cysteine residues with D_(H)2 could be found when the first cysteineof these CDR H3s was “fixed” prior to alignment. Four of the sevensequences (BLV1H12, BLV5D3, BLV8C11, and BF4E9) contain four cysteinesin the same positions as D_(H)2, but also have additional cysteines.BLV5B8 has two cysteines in common with the germline D_(H)2. Thislimited homology with some cysteine conservation suggests that mutationof D_(H)2 could generate these sequences. B-L1 and B-L2 are from initialsequences from bovine spleen, and the remaining are selected ultralongCDR H3 sequences from deep sequencing data. The first group contains thelongest CDR H3s identified, and appear clonally related. The * indicatesa sequence represented 167 times, suggesting it was strongly selectedfor function. Several of the eight-cysteine sequences appear selectedfor function as they were represented multiple times, indicated inparentheses. Other representative sequences of various lengths areindicated in the last group. The framework cysteine and tryptophanresidues that define the CDR H3 boundaries are double-underlined. Thesequences BLV1H12 through UL-77 (left-most column) presented in Tables2A-C are depicted broken apart into four segments to identify thesegments of amino acid residues that are derived from certain germlinesequences. Moving from left to right, the first segment is derived fromthe V_(H) germline and is represented throughout the disclosure as aX¹X²X³X⁴X⁵ motif. The second segment is a string of spacer amino acidresidues designated throughout the disclosure as X_(n) residues. Thethird segment is a string of amino acid residues derived from thegermline D_(H)2 region and the fourth segment is a string of amino acidresidues derived from the germline J_(H)1 region.

DETAILED DESCRIPTION

Disclosed herien are antibodies and fragments thereof. Generally, theantibodies and fragments thereof comprise at least a portion of anultralong CDR3. The portion of the ultralong CDR3 may be derived from orbased on an ultralong CDR3 sequence. The portion of the ultralong CDR3may be derived from or based on a stalk domain of an ultralong CDR3sequence. Alternatively, or additionally, the portion of the ultralongCDR3 may be derived from or based on a knob domain of an ultralong CDR3sequence. The antibodies and fragments thereof may further comprise oneor more therapeutic polypeptides. The therapeutic polypeptides may beinserted into the portion of the ultralong CDR3. The therapeuticpolypeptides may replace one or more amino acid residues in the aminoacid sequence of the portion of the ultralong CDR3. The therapeuticpolypeptides may replace one or more nucleotides in the nucleic acidsequence of the portion of the ultralong CDR3. Alternatively, thetherapeutic polypeptides may be conjugated or attached to the portion ofthe ultralong CDR3. The antibodies and fragments disclosed herein mayfurther comprise one or more linkers. Additionally, the antibodies andfragments disclosed herein further comprise a cleavage site. A portionof the antibodies and fragments disclosed herien may be based on orderived from an antibody sequence from a different animal or specie fromwith the ultralong CDR3 is derived. For example, the ultralong CDR3 maybe derived from or based on a bovine antibody sequence and theadditional and another portion of the antibody sequence may be derivedfrom or based on a non-bovine antibody sequence. Further details of theantibodies and fragments thereof are described herein.

Ultralong CDR3Proteins

The present disclosure provides antibodies or immunoglobulin constructscomprising ultralong CDR3 sequences or portions thereof.

In an embodiment, the present disclosure provides an antibody comprisingan ultralong CDR3. The ultralong CDR3 may be 35 amino acids in length ormore (e.g., 40 or more, 45 or more, 50 or more, 55 or more, 60 or more).The ultralong CDR3 may comprise at least a portion of a knob domain of aCDR3, at least a portion of a stalk domain of a CDR3, or a combinationthereof. The portion of the knob domain of the CDR3 may comprise one ormore conserved motifs derived from the knob domain of the ultralongCDR3. The portion of the stalk domain of the CDR3 may comprise one ormore conserved motis derived from the stalk domain of the ultralongCDR3. Such an antibody may comprise at least 3 cysteine residues or more(e.g., 4 or more, 6 or more, 8 or more) within the ultralong CDR3. Theantibody may comprise one or more cysteine motifs. The antibody maycomprise a non-antibody sequence within the ultralong CDR3.Alternatively, or additionally, the antibody comprises a non-bovinesequence. The non-bovine sequence can be linked to the ultralong CDR3sequence. The antibody may further comprise a linker. The linker cancomprise an amino acid sequence of (GGGGS)_(n) wherein n=1 to 5.Alternatively, the linker comprises an amino acids sequence of(GSG)_(n), GGGSGGGGS or GGGGSGGGS. The antibody may comprise anon-bovine sequence within the ultralong CDR3. The antibody may furthercomprise an antibody sequence, wherein the antibody sequence does notcomprise an ultralong CDR3 sequence. The antibody may further comprisean antibody sequence, wherein the amino acid sequence identitity of theantibody peptide sequence to the ultralong CDR3 peptide sequence isabout 40% or less (e.g., about 35% or less, about 30% or less, about 25%or less, about 20% or less, about 15% or less, 10% or less, about 5% orless, about 3% or less, about 1% or less). The antibody may comprise acytotoxic agent or therapeutic polypeptide. The cytotoxic agent ortherapeutic polypeptide may be conjugated to the ultralong CDR3. Theantibody may bind to a target. The target may be a protein target. Theprotein target may be a transmembrane protein target. The antibody maycomprise at least a portion of a BLV1H12 and/or BLVCV1 antibody.Alternatively, or additionally, the antibody comprises at least aportion of a BLV5D3, BLV8C11, BF1H1, BLV5B8 and/or F18 antibody. Theantibody may comprise at least a portion of a human antibody. Theantibody may be a chimeric, recombinant, engineered, synthetic,humanized, fully human, or human engineered antibody. The antibody maycomprise antibody sequences from two or more different antibodies. Thetwo or more different antibodies may be from the same species. Forexample, the specie may be a bovine specie, human specie, or murinespecie. The two or more different antibodies may be from the same typeof animal. For example the two or more different antibodies may be froma cow. The two or more different antibodies may be from a human.Alternatively, the two or more different antibodies are from differentspecies. For example, the two or more different antibodies are from ahuman specie and bovine specie. In another example, the two or morediffent antibodies are from a bovine specie and a non-bovine specie. Inanother example, the two or more different antibodies are from a humanspecie and a non-human specie.

In another embodiment, the present disclosure provides an antibodycomprising an ultralong CDR3, wherein the CDR3 is 35 amino acids inlength or more and is derived from or based on a non-human sequence. Theultralong CDR3 sequence may be derived from any species that naturallyproduces ultralong CDR3 antibodies, including ruminants such as cattle(Bos taurus). The antibody may comprise at least a portion of a BLV1H12and/or BLVCV1 antibody. Alternatively, or additionally, the antibodycomprises at least a portion of a BLV5D3, BLV8C11, BF1H1, BLV5B8 and/orF18 antibody. Alternatively, the ultralong CDR3 sequence may be derivedfrom a camelid or shark CDR3 sequence.

In another embodiment, the present disclosure provides an antibodycomprising an ultralong CDR3, wherein the CDR3 comprises a non-antibodysequence. The non-antibody sequence may be derived from any proteinfamily including, but not limited to, chemokines, growth factors,peptides, cytokines, cell surface proteins, serum proteins, toxins,extracellular matrix proteins, clotting factors, secreted proteins, etc.The non-antibody sequence may be derived from a therapeutic polypeptide.The non-antibody sequence may be of human or non-human origin. Thenon-antibody sequence may comprise a synthetic sequence. Thenon-antibody sequence may comprise a portion of a non-antibody proteinsuch as a peptide or domain. The non-antibody sequence of an ultralongCDR3 may contain mutations from its natural sequence, including aminoacid changes (e.g., substitutions), insertions or deletions. Engineeringadditional amino acids at the junction between the non-antibody sequencemay be done to facilitate or enhance proper folding of the non-antibodysequence within the antibody. The CDR3 may be 35 amino acids in lengthor more. The ultralong CDR3 may comprise at least a portion of a knobdomain of a CDR3, at least a portion of a stalk domain of a CDR3, or acombination thereof. The portion of the knob domain of the CDR3 maycomprise one or more conserved motifs derived from the knob domain ofthe ultralong CDR3. The portion of the stalk domain of the CDR3 maycomprise one or more conserved motis derived from the stalk domain ofthe ultralong CDR3. Alternatively, or additionally, the antibodycomprises at least 3 cysteine residues or more. The antibody cancomprise one or more cysteine motifs. The antibody may comprise at leasta portion of a BLV1H12 and/or BLVCV1 antibody. Alternatively, oradditionally, the antibody comprises at least a portion of a BLV5D3,BLV8C11, BF1H1, BLV5B8 and/or F18 antibody.

In another embodiment, the present disclosure provides an antibodycomprising an ultralong CDR3 and a non-bovine sequence. The ultralongCDR3 can be derived from a ruminant. The ruminant can be a bovine. Thenon-bovine sequence can be derived from or based on a non-bovine mammalsequence. For example, the non-bovine sequence can be derived from orbased on a human, mouse, rat, sheep, dog, and/or goat sequence. Theultralong CDR3 sequence can comprise the non-bovine sequence.Alternatively, the non-bovine sequence is linked to the ultralong CDR3sequence. The non-bovine sequence can be derived from or based on atleast a portion of an antibody sequence. The antibody sequence canencode a variable region, constant region or a combination thereof. TheCDR3 may be 35 amino acids in length or more. The ultralong CDR3 maycomprise at least a portion of a knob domain of a CDR3, at least aportion of a stalk domain of a CDR3, or a combination thereof. Theportion of the knob domain of the CDR3 may comprise one or moreconserved motifs derived from the knob domain of the ultralong CDR3. Theportion of the stalk domain of the CDR3 may comprise one or moreconserved motis derived from the stalk domain of the ultralong CDR3.Alternatively, or additionally, the antibody comprises at least 3cysteine residues or more. The antibody can comprise one or morecysteine motifs.

In another embodiment, the present disclosure provides an antibodycomprising an ultralong CDR3, wherein the CDR3 is 35 amino acids inlength or more and comprises at least 3 cysteine residues or more,including, for example, 4 or more, 6 or more, and 8 or more.

In another embodiment, the present disclosure provides for an antibodycomprising an ultralong CDR3 wherein the CDR3 is 35 amino acids inlength or more and comprises at least 3 cysteine residues or more andwherein the ultralong CDR3 is a component of a multispecific antibody.The multispecific antibody may be bispecific or comprise greatervalencies.

In another embodiment, the present disclosure provides an antibodycomprising an ultralong CDR3, wherein the CDR3 is 35 amino acids inlength or more and comprises at least 3 cysteine residues or more,wherein the ultralong CDR3 is a component of an immunoconjugate.

In another embodiment, the present disclosure provides an antibodycomprising an ultralong CDR3, wherein the CDR3 is 35 amino acids inlength or more and comprises at least 3 cysteine residues or more,wherein the antibody comprising an ultralong CDR3 binds to atransmembrane protein target. Such transmembrane targets may include,but are not limited to, GPCRs, ion channels, transporters, and cellsurface receptors.

In another embodiment, the present disclosure provides an antibodycomprising an ultralong CDR3, wherein the antibody comprising anultralong CDR3 binds to a transmembrane protein target. Suchtransmembrane targets may include, but are not limited to, GPCRs, ionchannels, transporters, and cell surface receptors. The CDR3 may be 35amino acids in length or more. The ultralong CDR3 may comprise at leasta portion of a knob domain of a CDR3, at least a portion of a stalkdomain of a CDR3, or a combination thereof. The portion of the knobdomain of the CDR3 may comprise one or more conserved motifs derivedfrom the knob domain of the ultralong CDR3. The portion of the stalkdomain of the CDR3 may comprise one or more conserved motis derived fromthe stalk domain of the ultralong CDR3. Alternatively, or additionally,the antibody comprises at least 3 cysteine residues or more. Theantibody can comprise one or more cysteine motifs.

Provided herein is an immunoglobulin construct comprising a mammalianimmunoglobulin heavy chain comprising at least a portion ofcomplementarity-determining region 3 (CDR3H); and a therapeuticpolypeptide, wherein the therapeutic polypeptide is inserted into orreplaces at least a portion of the CDR3H. The immunoglobulin constructmay comprise one or more linkers. The one or more linkers can connectthe therapeutic polypeptide to the heavy chain. In some embodiments, thelinker comprises an amino acid sequence of (GGGGS)n wherein n=1 to 5.Alternatively, or additionally, the linker comprises an amino acid acidsequence of (GSG)n (SEQ ID NO: 342), GGGSGGGGS (SEQ ID NO: 337) orGGGGSGGGS. In some embodiments provided are immunoglobulin constructsdescribed herein, wherein the therapeutic polypeptide is selected from ahormone, a lymphokine, an interleukin, a chemokines, a cytokine andcombinations thereof. In certain embodiments, the therapeuticpolypeptide is a cytokine. In some embodiments, the therapeuticpolypeptide is a colony stimulating factor polypeptide. In certainembodiments, the colony stimulating factor is macrophagecolony-stimulating factor (M-C SF), Granulocyte-macrophagecolony-stimulating factor (GM-CSF), Granulocyte colony-stimulatingfactor (G-CSF) or fragment, or variant thereof. In an embodiment, thecolony stimulating factor is mammalian G-CSF or derivative or variantthereof. In a certain embodiment, the colony stimulating factor isbovine G-CSF or derivative or variant thereof. In other instances, thetherapeutic polypeptide is Moka1, Vm24, human GLP-1, Exendin-4, humanEPO, human FGF21, human GMCSF, or human interferon-beta. Provided hereinare immunoglobulin constructs comprising a mammalian immunoglobulinheavy chain comprising a knob domain in the complementarity-determiningregion 3 (CDR3H) or fragment thereof; and a therapeutic polypeptideattached to said knob domain of the CDR3H, wherein said mammalianimmunoglobulin is a bovine immunoglobulin. In some embodiments, thebovine immunoglobulin is a BLV1H12 antibody. In some embodiments of theimmunoglobulin constructs described herein, at least a portion of theknob domain is replaced by the therapeutic polypeptide. The knob domainof the CDR3H may comprise one or more conserved motifs derived from theknob domain of the ultralong CDR3H. The immunoglobulin construct mayfurther comprise at least a portion of a stalk domain in the CDR3H. Theportion of the stalk domain of the CDRH3 may comprise one or moreconserved motis derived from the stalk domain of the ultralong CDR33H.

Further provided herein are antibodies or fragments thereof comprising astalk domain in the complementarity-determining region 3 (CDR3H) orfragment thereof; and a therapeutic polypeptide. In some instances, thecomplementarity-determining region 3 (CDR3H) is derived from a bovineultralong CDR3H. The therapeutic polypeptide can be any of thetherapeutic polypeptides disclosed herein. For example, the therapeuticpolypeptide is Moka1, Vm24, GLP-1, Exendin-4, human EPO, human FGF21,human GMCSF, or human interferon-beta. The therapeutic polypeptide canbe attached to the stalk domain. In some instances, the antibody orfragment thereof further comprises a linker. The linker can attach thetherapeutic polypeptide to the stalk domain. Alternatively, oradditionally, the antibody or fragment thereof further comprises atleast a portion of a knob domain in the CDR3H. In some instances, thelinker attaches the therapeutic polypeptide to the knob domain. In someinstances, the knob domain is attached to the stalk domain. The portionof the knob domain of the CDR3 may comprise one or more conserved motifsderived from the knob domain of the ultralong CDR3. The stalk domain ofthe CDR3 may comprise one or more conserved motis derived from the stalkdomain of the ultralong CDR3.

In some instances, an antibody or fragment thereof is provided herein.The antibody or fragment thereof can comprise at least oneimmunoglobulin domain or fragment thereof; and a therapeutic polypeptideor derivative or variant thereof. The therapeutic polypeptide, orderivative or variant thereof can be attached to the immunoglobulindomain. In some instances, the therapeutic polypeptide is Moka1, Vm24,GLP-1, Exendin-4, human EPO, human FGF21, human GMCSF, humaninterferon-beta, or derivative or variant thereof. In some embodiments,the immunoglobulin domain is an immunoglobulin A, an immunoglobulin D,an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. Theimmunoglobulin domain can be an immunoglobulin heavy chain region orfragment thereof. In some instances, the immunoglobulin domain is from amammalian antibody. Alternatively, the immunoglobulin domain is from achimeric antibody. In some instances, the immunoglobulin domain is froman engineered antibody or recombinant antibody. In other instances, theimmunoglobulin domain is from a humanized, human engineered or fullyhuman antibody. In certain embodiments, the mammalian antibody is abovine antibody. In other instances, the mammalin antibody is a humanantibody. In other instances, the mammalian antibody is a murineantibody. In some instances, the immunoglobulin domain is a heavy chainregion comprising a knob domain in the complementarity-determiningregion 3 (CDR3H) or fragment thereof. The therapeutic polypeptide can beattached to the knob domain. Alternatively, or additionally, theimmunoglobulin domain is a heavy chain region comprising a stalk domainin the complementarity-determining region 3 (CDR3H) or fragment thereof.In some instances, the therapeutic polypeptide is attached to the stalkdomain. In some instances, the antibody or fragment thereof furthercomprises a linker. The linker can attach the therapeutic polypeptide tothe immunoglobulin domain or fragment thereof. The knob domain of theCDR3 may comprise one or more conserved motifs derived from the knobdomain of the ultralong CDR3. The stalk domain of the CDR3 may compriseone or more conserved motis derived from the stalk domain of theultralong CDR3.

Provided herein is an immunoglobulin construct comprising at least oneimmunoglobulin domain or fragment thereof; and a G-CSF polypeptide orderivative or variant thereof attached to said immunoglobulin domain. Insome embodiments, the immunoglobulin domain is an immunoglobulin A, animmunoglobulin D, an immunoglobulin E, an immunoglobulin G, or animmunoglobulin M. In some embodiments, the immunoglobulin domain is animmunoglobulin heavy chain region or fragment thereof. In an embodiment,the immunoglobulin domain is from a mammalian or chimeric antibody. Inother instances, the immunoglobulin domain is from a humanized, humanengineered or fully human antibody. In certain embodiments, themammalian antibody is a bovine antibody. In some instances, themammalian antibody is a human antibody. In other instances, themammalian antibody is a murine antibody. In an embodiment, theimmunoglobulin domain is a heavy chain region comprising a knob domainin the complementarity-determining region 3 (CDR3H) or fragment thereof.In an embodiment, the G-CSF polypeptide is attached to the knob domain.The immunoglobulin domain may be a heavy chain region comprising a stalkdomain in the complementarity-determining region 3 (CDR3H) or fragmentthereof. The G-CSF polypeptide may be attached to the stalk domain. Theknob domain of the CDR3 may comprise one or more conserved motifsderived from the knob domain of the ultralong CDR3. The stalk domain ofthe CDR3 may comprise one or more conserved motis derived from the stalkdomain of the ultralong CDR3.

In certain embodiments, provided is an immunoglobulin constructcomprising at least one immunoglobulin domain or fragment thereof; and aG-CSF polypeptide or derivative or variant thereof attached to saidimmunoglobulin domain, wherein said G-CSF polypeptide is a bovine G-CSFpolypeptide or derivative or variant thereof. In certain embodimentsprovided herein is a pharmaceutical composition comprising animmunoglobulin construct provided herein, and a pharmaceuticallyacceptable carrier. In certain embodiments is provided a method ofpreventing or treating a disease in a mammal in need thereof comprisingadministering a pharmaceutical composition described herein to saidmammal. The immunoglobulin domain may be a heavy chain region comprisinga knob domain in the complementarity-determining region 3 (CDR3H) orfragment thereof. The G-CSF polypeptide may be attached to the knobdomain. The immunoglobulin domain may be a heavy chain region comprisinga stalk domain in the complementarity-determining region 3 (CDR3H) orfragment thereof. The G-CSF polypeptide may be attached to the stalkdomain. The knob domain of the CDR3 may comprise one or more conservedmotifs derived from the knob domain of the ultralong CDR3. The stalkdomain of the CDR3 may comprise one or more conserved motis derived fromthe stalk domain of the ultralong CDR3.

In some embodiments is an antibody or fragment thereof comprising: (a) afirst antibody sequence, wherein at least a portion of the firstantibody sequence is derived from at least a portion of an ultralongCDR3; and (b) a non-antibody sequence. The antibody or fragment thereofmay further comprise a second antibody sequence, wherein at least aportion of the second antibody sequence is derived from at least aportion of an ultralong CDR3. The ultralong CDR3 from which the firstantibody sequence and/or second antibody sequence may be derived from aruminant. The ruminant can be a cow. At least a portion of the firstantibody sequence and/or at least a portion of the second antibodysequence can be derived from a mammal. The mammal may be a bovine.Alternatively, the mammal is a non-bovine mammal, such as a human. Thefirst and/or second antibody sequences may be 3 or more amino acids inlength. The amino acids may be consecutive amino acids. Alternatively,the amino acids are non-consecutive amino acids. The first and/or secondantibody sequences may comprise a bovine antibody sequence comprising 3or more amino acids in length. The bovine antibody may be a BLVH12,BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 antibody. The firstand/or second antibody sequences may comprise a human antibody sequencecomprising 3 or moreore amino acids in length. The portion of the firstantibody sequence derived from at least a portion of the ultralong CDR3and/or the portion of the second antibody sequence derived from at leasta portion of the ultralong CDR3 can be 20 or fewer amino acids inlength. The portion of the first antibody sequence derived from at leasta portion of the ultralong CDR3 and/or the portion of the secondantibody sequence derived from at least a portion of the ultralong CDR3may be 3 or more amino acids in length. The first and/or second antibodysequences can comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 ormore, 20 or more, 30 or more, or 40 or more amino acid residues derivedfrom a knob domain of the ultralong CDR3. The 1 or more amino acidresidues derived from the knob domain of the ultralong CDR3 may be aserine and/or cysteine residue. The first and/or second antibodysequences may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more aminoacid residues derived from a stalk domain of the ultralong CDR3. Thefirst and/or second antibody sequences may comprise 1 or more, 2 ormore, 3 or more, 4 or more, or 5 or more conserved motifs derived from astalk domain of the ultralong CDR3. The one or more conserved motifsderived from the stalk domain of the ultralong CDR3 may comprise asequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS:333-336. The portion of the first antibody sequence derived from atleast a portion of the ultralong CDR3 and/or the portion of the secondantibody sequence derived from at least a portion of the ultralong CDR3may comprise a sequence selected from any one of SEQ ID NOS: 157-307 andSEQ ID NOS: 333-336. The portion of the first antibody sequence derivedfrom at least a portion of the ultralong CDR3 and/or the portion of thesecond antibody sequence derived from at least a portion of theultralong CDR3 may comprise a sequence that is 50% or more homologous toa sequence selected from any one of SEQ ID NOS: 157-224 and 235-295. Theportion of the first antibody sequence derived from at least a portionof the ultralong CDR3 may comprise a sequence selected from any one ofSEQ ID NOS: 157-234. The portion of the first antibody sequence derivedfrom at least a portion of the ultralong CDR3 may comprise a sequencethat is 50% or more homologous to a sequence selected from any one ofSEQ ID NOS: 157-224. The portion of the first antibody sequence derivedfrom at least a portion of the ultralong CDR3 may comprise a sequencethat is 50% or more homologous to a sequence selected from any one ofSEQ ID NOS: 225-227. The portion of the second antibody sequence derivedfrom at least a portion of the ultralong CDR3 may comprise a sequenceselected from any one of SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.The portion of the second antibody sequence derived from at least aportion of the ultralong CDR3 may comprise a sequence that may be 50% ormore homologous to a sequence selected from any one of SEQ ID NOS:235-295. The portion of the first antibody sequence derived from atleast a portion of the ultralong CDR3 and the portion of the secondantibody sequence derived from at least a portion of the ultralong CDR3may be derived from the same ultralong CDR3 sequence. The portion of thefirst antibody sequence derived from at least a portion of the ultralongCDR3 and the portion of the second antibody sequence derived from atleast a portion of the ultralong CDR3 may be derived from two or moredifferent ultralong CDR3 sequences. The portions of the ultralong CDR3of the first and/or second antibody sequences may be based on or derivedfrom a BLV1H12 ultralong CDR3 sequence. The portions of the ultralongCDR3 of the first and/or second antibody sequences may be based on orderived from a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralongCDR3 sequence. The antibody may further comprise one or more linkersequences.

The present disclosure also provides antibodies that comprise a heavychain polypeptide, wherein the heavy chain polypeptide comprises atleast a portion of an ultralong CDR3 sequence. The heavy chainpolypeptide may comprise a polypeptide sequence based on or derived froma polypeptide sequence of any one of SEQ ID NOS: 24-44. The heavy chainpolypeptide may comprise a polypeptide sequence encoded by a DNAsequence based on or derived from the DNA sequence of any one of SEQ IDNOS: 2-22. Also provided are antibodies comprising a heavy chainpolypeptide, wherein the heavy chain polypeptide comprises an ultralongCDR3 sequence and the heavy chain polypeptide sequences aresubstantially similar to those polypeptide sequences provided by any oneof SEQ ID NOS: 24-44. A heavy chain polypeptide sequence may beconsidered substantially similar to a polypeptide sequence provided byany one of SEQ ID NOS: 24-44 where the heavy chain polypeptide sequenceshares 60%, 70%, 80%, 90%, 95%, 99%, or more nucleic acid identity to anucleotide sequence provided by any one of SEQ ID NOS: 24-44. Theantibodies may further comprise a light chain polypeptide. The lightchain polypeptide may comprise a polypeptide sequence based on orderived from a polypeptide sequence of SEQ ID NO: 23. The light chainpolypeptide may comprise a polypeptide sequence encoded by a DNAsequence based on or derived from the DNA sequence of SEQ ID NO: 1. Alsoprovided are antibodies further comprising a light chain polypeptide,wherein the light chain polypeptide comprises an ultralong CDR3 sequenceand the light chain polypeptide sequences are substantially similar tothose polypeptide sequences provided by SEQ ID NO: 23. A light chainpolypeptide sequence may be considered substantially similar to apolypeptide sequence provided by SEQ ID NO: 1 where the light chainpolypeptide sequence shares 60%, 70%, 80%, 90%, 95%, 99%, or morenucleic acid identity to a nucleotide sequence provided by any one ofSEQ ID NO: 1. The antibody may have therapeutic activity in an animal.The antibody can have therapeutic activity in infectious disease in asubject. The antibody may comprise a monoclonal antibody, polyclonalantibody, chimeric antibody, recombinant antibody, engineered antibody,or synthetic antibody. The antibody may comprise a mammalian antibody.The antibody may comprise a bovine antibody. The antibody may comprise aG-CSF polypeptide, or derivative or variant thereof. The antibody maycomprise a mammalian G-CSF polypeptide, or derivative or variantthereof. The antibody may comprise a bovine G-CSF, or derivative orvariant thereof. In some embodiments, a pharmaceutical composition oftherapeutic formulation comprises an antibody described herein and apharmaceutically acceptable carrier. In certain embodiments, theantibody is used in a method of treating a subject in need thereof, witha therapeutically effective amount of the antibody or a pharmaceuticalcomposition described herein. In some embodiments, a nucleic acidmolecule or a complement thereof encodes a therapeutic immunoglobulindescribed herein.

Genetic Sequences

The present disclosure provides genetic sequences (e.g., genes, nucleicacids, polynucleotides) encoding antibodies comprising ultralong CDR3sequences or portions thereof. The present disclosure provides geneticsequences (e.g., genes, nucleic acids, polynucleotides) encodingantibodies comprising the knob domain and/or knob domain of ultralongCDR3 sequences. In another embodiment, the present disclosure providesgenetic sequences encoding an antibody or immunoglobulin constructdescribed herein.

The present disclosure also provides genetic sequences (e.g., genes,nucleic acids, polynucleotides) encoding an ultralong CDR3 or portionthereof. The present disclosure also provides genetic sequences (e.g.,genes, nucleic acids, polynucleotides) encoding the knob domain and/orknob domain of an ultralong CDR3.

In an embodiment, the present disclosure provides genetic sequencesencoding an antibody comprising an ultralong CDR3. The ultralong CDR3may be 35 amino acids in length or more (e.g., 40 or more, 45 or more,50 or more, 55 or more, 60 or more). The ultralong CDR3 may comprise atleast a portion of a knob domain of a CDR3, at least a portion of astalk domain of a CDR3, or a combination thereof. Such an antibody maycomprise at least 3 cysteine residues or more (e.g., 4 or more, 6 ormore, 8 or more) within the ultralong CDR3. The antibody may compriseone or more cysteine motifs. The antibody may comprise a non-antibodysequence within the ultralong CDR3. Alternatively, or additionally, theantibody comprises a non-bovine sequence. The antibody may furthercomprise an antibody sequence. The antibody may comprise a cytotoxicagent or therapeutic polypeptide. The cytotoxic agent or therapeuticpolypeptide may be conjugated to the ultralong CDR3. The antibody maybind to a target. The target may be a protein target, such as atransmembrane protein target.

In another embodiment, the present disclosure provides genetic sequencesencoding an antibody comprising an ultralong CDR3, wherein the CDR3 is35 amino acids in length or more and is derived from or based on anon-human sequence. The genetic sequences encoding the ultralong CDR3may be derived from any species that naturally produces ultralong CDR3antibodies, including ruminants such as cattle (Bos taurus).Alternatively, the ultralong CDR3 sequence may be derived from a camelidor shark CDR3 sequence.

In another embodiment, the present disclosure provides genetic sequencesencoding an antibody comprising an ultralong CDR3, wherein the CDR3comprises a non-antibody protein sequence. The genetic sequencesencoding the non-antibody protein sequences may be derived from anyprotein family including, but not limited to, chemokines, growthfactors, peptides, cytokines, cell surface proteins, serum proteins,toxins, extracellular matrix proteins, clotting factors, secretedproteins, etc. The non-antibody sequence may be derived from atherapeutic polypeptide. The non-antibody protein sequence may be ofhuman or non-human origin. The non-antibody sequence may comprise asynthetic sequence. The non-antibody sequencemay comprise a portion of anon-antibody protein such as a peptide or domain. The non-antibodyprotein sequence of an ultralong CDR3 may contain mutations from itsnatural sequence, including amino acid changes (e.g., substitutions),insertions or deletions. Engineering additional amino acids at thejunction between the non-antibody sequence may be done to facilitate orenhance proper folding of the non-antibody sequence within the antibody.The CDR3 may be 35 amino acids in length or more. The ultralong CDR3 maycomprise at least a portion of a knob domain of a CDR3, at least aportion of a stalk domain of a CDR3, or a combination thereof.Alternatively, or additionally, the antibody comprises at least 3cysteine residues or more. The antibody can comprise one or morecysteine motifs.

In another embodiment, the present disclosure provides genetic sequencesencoding an antibody comprising an ultralong CDR3 and a non-bovinesequence. The ultralong CDR3 can be derived from a ruminant. Theruminant can be a bovine. The non-bovine sequence can be derived from orbased on a non-bovine mammal sequence. For example, the non-bovinesequence can be derived from or based on a human, mouse, rat, sheep,dog, and/or goat sequence. The non-bovine sequence can be within theultralong CDR3. Alternatively, the non-bovine sequence is linked orattached to the ultralong CDR3 sequence. The non-bovine sequence can bederived from or based on at least a portion of an antibody sequence. Theantibody sequence can encode a variable region, constant region or acombination thereof. The CDR3 may be 35 amino acids in length or more.The ultralong CDR3 may comprise at least a portion of a knob domain of aCDR3, at least a portion of a stalk domain of a CDR3, or a combinationthereof. Alternatively, or additionally, the antibody comprises at least3 cysteine residues or more. The antibody can comprise one or morecysteine motifs.

In another embodiment, the present disclosure provides genetic sequencesencoding an antibody comprising an ultralong CDR3, wherein the CDR3 is35 amino acids in length or more and comprises at least 3 cysteineresidues or more, including, for example, 4 or more, 6 or more, and 8 ormore.

In another embodiment, the present disclosure provides genetic sequencesencoding an antibody comprising an ultralong CDR3 wherein the CDR3 is 35amino acids in length or more and comprises at least 3 cysteine residuesor more and wherein the ultralong CDR3 is a component of a multispecificantibody. The multispecific antibody may be bispecific or comprisegreater valencies.

In another embodiment, the present disclosure provides genetic sequencesencoding an antibody comprising an ultralong CDR3, wherein the CDR3 is35 amino acids in length or more and comprises at least 3 cysteineresidues or more, wherein the ultralong CDR3 is a component of animmunoconjugate.

In another embodiment, the present disclosure provides genetic sequencesencoding an antibody comprising an ultralong CDR3 wherein the CDR3 is 35amino acids in length or more and comprises at least 3 cysteine residuesor more and wherein the antibody comprising an ultralong CDR3 binds to atransmembrane protein target. Such transmembrane targets may include,but are not limited to, GPCRs, ion channels, transporters, and cellsurface receptors.

In another embodiment, the present disclosure provides genetic sequencesencoding an antibody comprising an ultralong CDR3, wherein the antibodycomprising an ultralong CDR3 binds to a transmembrane protein target.Such transmembrane targets may include, but are not limited to, GPCRs,ion channels, transporters, and cell surface receptors. The CDR3 may be35 amino acids in length or more. The ultralong CDR3 may comprise atleast a portion of a knob domain of a CDR3, at least a portion of astalk domain of a CDR3, or a combination thereof. Alternatively, oradditionally, the antibody comprises at least 3 cysteine residues ormore. The antibody can comprise one or more cysteine motifs.

In another embodiment, the present disclosure provides genetic sequencesencoding an antibody or fragment thereof comprising: (a) a firstantibody sequence, wherein at least a portion of the first antibodysequence is derived from at least a portion of an ultralong CDR3; and(b) a non-antibody sequence. The antibody or fragment thereof mayfurther comprise a second antibody sequence, wherein at least a portionof the second antibody sequence is derived from at least a portion of anultralong CDR3. The ultralong CDR3 from which the first antibodysequence and/or second antibody sequence may be derived from a ruminant.The ruminant can be a cow. At least a portion of the first antibodysequence and/or at least a portion of the second antibody sequence canbe derived from a mammal. The mammal may be a bovine. Alternatively, themammal is a non-bovine mammal, such as a human. The first and/or secondantibody sequences may be 3 or more amino acids in length. The aminoacids may be consecutive amino acids. Alternatively, the amino acids arenon-consecutive amino acids. The first and/or second antibody sequencesmay comprise a bovine antibody sequence comprising 3 or more amino acidsin length. The bovine antibody may be a BLVH12, BLV5B8, BLVCV1, BLV5D3,BLV8C11, BF1H1, or F18 antibody. The first and/or second antibodysequences may comprise a human antibody sequence comprising 3 or more, 4or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 ormore, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 ormore, or 70 or more amino acids in length. The portion of the firstantibody sequence derived from at least a portion of the ultralong CDR3and/or the portion of the second antibody sequence derived from at leasta portion of the ultralong CDR3 can be 20 or fewer amino acids inlength. The portion of the first antibody sequence derived from at leasta portion of the ultralong CDR3 and/or the portion of the secondantibody sequence derived from at least a portion of the ultralong CDR3may be 3 or more amino, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 15 or more, or 20 or more acids in length.The first and/or second antibody sequences can comprise 1 or more, 2 ormore, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more,9 or more, 10 or more, 15 or more, 20 or more, 30 or more, or 40 or moreamino acid residues derived from a knob domain of the ultralong CDR3.The 1 or more amino acid residues derived from the knob domain of theultralong CDR3 may be a serine and/or cysteine residue. The first and/orsecond antibody sequences may comprise 1 or more, 2 or more, 3 or more,4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10or more amino acid residues derived from a stalk domain of the ultralongCDR3. The first and/or second antibody sequences may comprise 1 or more,2 or more, 3 or more, 4 or more, or 5 or more conserved motifs derivedfrom a stalk domain of the ultralong CDR3. The one or more conservedmotifs derived from the stalk domain of the ultralong CDR3 may comprisea sequence selected from any one of SEQ ID NOS: 157-307 and SEQ ID NOS:333-336. The portion of the first antibody sequence derived from atleast a portion of the ultralong CDR3 and/or the portion of the secondantibody sequence derived from at least a portion of the ultralong CDR3may comprise a sequence selected from any one of SEQ ID NOS: 157-307 andSEQ ID NOS: 333-336. The portion of the first antibody sequence derivedfrom at least a portion of the ultralong CDR3 and/or the portion of thesecond antibody sequence derived from at least a portion of theultralong CDR3 may comprise a sequence that is 50% or more homologous toa sequence selected from any one of SEQ ID NOS: 157-224 and 235-295. Theportion of the first antibody sequence derived from at least a portionof the ultralong CDR3 may comprise a sequence selected from any one ofSEQ ID NOS: 157-234. The portion of the first antibody sequence derivedfrom at least a portion of the ultralong CDR3 may comprise a sequencethat is 50% or more homologous to a sequence selected from any one ofSEQ ID NOS: 157-224. The portion of the first antibody sequence derivedfrom at least a portion of the ultralong CDR3 may comprise a sequencethat is 50% or more homologous to a sequence selected from any one ofSEQ ID NOS: 225-227. The portion of the second antibody sequence derivedfrom at least a portion of the ultralong CDR3 may comprise a sequenceselected from any one of SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336.The portion of the second antibody sequence derived from at least aportion of the ultralong CDR3 may comprise a sequence that may be 50% ormore homologous to a sequence selected from any one of SEQ ID NOS:235-295. The portion of the first antibody sequence derived from atleast a portion of the ultralong CDR3 and the portion of the secondantibody sequence derived from at least a portion of the ultralong CDR3may be derived from the same ultralong CDR3 sequence. The portion of thefirst antibody sequence derived from at least a portion of the ultralongCDR3 and the portion of the second antibody sequence derived from atleast a portion of the ultralong CDR3 may be derived from two or moredifferent ultralong CDR3 sequences. The portions of the ultralong CDR3of the first and/or second antibody sequences may be based on or derivedfrom a BLV1H12 ultralong CDR3 sequence. The portions of the ultralongCDR3 of the first and/or second antibody sequences may be based on orderived from a BLV5B8, BLVCV1, BLV5D3, BLV8C11, BF1H1, or F18 ultralongCDR3 sequence. The antibody may further comprise one or more linkersequences.

The present disclosure also provides isolated genetic sequences (e.g.,genes, nucleic acids, polynucleotides, oligonucleotides) such as geneticsequences encoding antibodies that comprise an ultralong CDR sequenceincluding, for example, a CDR3 sequence as provided by any one of SEQ IDNOS: 2-22. Also provided are ultralong CDR3 nucleic acid sequences thatare substantially similar to those CDR3 sequences provided by any one ofSEQ ID NOS: 2-22. A CDR3 sequence may be considered substantiallysimilar to a CDR3 sequence provided by any one of SEQ ID NOS: 2-22 wherethe CDR3 sequence shares 80%, 85%, 90%, 95%, 99%, or more, nucleic acidsequence identity to a CDR3 sequence provided by any one of SEQ ID NOS:2-22 or hybridizes to any one of SEQ ID NOS: 2-22 under stringenthybridization conditions.

The present disclosure also provides isolated genetic sequences (e.g.,genes, nucleic acids, polynucleotides, oligonucleotides) such as geneticsequences encoding antibodies that comprise a heavy chain polypeptide,wherein the heavy chain polypeptide comprises at least a portion of anultralong CDR3 sequence. The heavy chain polypeptide may comprise apolypeptide sequence of any one of SEQ ID NOS: 24-44. The heavy chainpolypeptide may comprise a polypeptide sequence encoded by the DNA ofany one of SEQ ID NOS: 2-22. Also provided are isolated geneticsequences (e.g., genes, nucleic acids, polynucleotides,oligonucleotides) such as genetic sequences encoding antibodiescomprising a heavy chain polypeptide, wherein the heavy chainpolypeptide comprises an ultralong CDR3 sequence and the heavy chainpolypeptide sequences are substantially similar to those polypeptidesequences provided by any one of SEQ ID NOS: 24-44. A heavy chainpolypeptide sequence may be considered substantially similar to apolypeptide sequence provided by any one of SEQ ID NOS: 24-44 where theheavy chain polypeptide sequence shares 60%, 70%, 80%, 90%, 95%, 99%, ormore nucleic acid identity to a nucleotide sequence provided by any oneof SEQ ID NOS: 24-44 or hybridizes to any one of SEQ ID NOS: 24-44 understringent hybridization conditions. The isolated genetic sequences(e.g., genes, nucleic acids, polynucleotides, oligonucleotides) such asgenetic sequences encoding antibodies may further comprise a light chainpolypeptide. The light chain polypeptide may comprise a polypeptidesequence of SEQ ID NO: 23. The light chain polypeptide may comprise apolypeptide sequence encoded by the DNA of SEQ ID NO: 1. Also providedare isolated genetic sequences (e.g., genes, nucleic acids,polynucleotides, oligonucleotides) such as genetic sequences encodingantibodies further comprising a light chain polypeptide, wherein thelight chain polypeptide comprises an ultralong CDR3 sequence and thelight chain polypeptide sequences are substantially similar to thosepolypeptide sequences provided by SEQ ID NO: 23. A light chainpolypeptide sequence may be considered substantially similar to apolypeptide sequence provided by SEQ ID NO: 1 where the light chainpolypeptide sequence shares 60%, 70%, 80%, 90%, 95%, 99%, or morenucleic acid identity to a nucleotide sequence provided by any one ofSEQ ID NO: 1 or hybridizes to SEQ ID NOS: 1 under stringenthybridization conditions.

Libraries and Arrays

The present disclosure provides collections, libraries and arrays ofantibodies comprising ultralong CDR3 sequences. In some embodiments,members of the collections, libraries, or arrays may exhibit sequencediversity.

In an embodiment, the present disclosure provides a library or an arrayof antibodies comprising ultralong CDR3 sequences wherein at least twomembers of the library or array differ in the positions of at least oneof the cysteines in the ultralong CDR3 sequence. Structural diversitymay be enhanced through different numbers of cysteines in the ultralongCDR3 sequence (e.g., at least 3 or more cysteine residues such as 4 ormore, 6 or more and 8 or more) and/or through different disulfide bondformation, and hence different loop structures.

In another embodiment, the present disclosure provides for a library oran array of antibodies comprising ultralong CDR3 sequences wherein atleast two members of the library or the array differ in at least oneamino acid located between cysteines in the ultralong CDR3. In thisregard, members of the library or the array can contain cysteines in thesame positions of CDR3, resulting in similar overall structural folds,but with fine differences brought about through different amino acidside chains. Such libraries or arrays may be useful for affinitymaturation.

In another embodiment, the present disclosure provides libraries orarrays of antibodies comprising ultralong CDR3 sequences wherein atleast two of the ultralong CDR3 sequences differ in length (e.g., 35amino acids in length or more such as 40 or more, 45 or more, 50 ormore, 55 or more and 60 or more). The amino acid and cysteine contentmay or may not be altered between the members of the library or thearray. Different lengths of ultralong CDR3 sequences may provide forunique binding sites, including, for example, due to steric differences,as a result of altered length.

In another embodiment, the present disclosure provides libraries orarrays of antibodies comprising ultralong CDR3 sequences wherein atleast two members of the library differ in the human framework used toconstruct the antibody comprising an ultralong CDR3.

In another embodiment, the present disclosure provides libraries orarrays of antibodies comprising ultralong CDR3 sequences wherein atleast two members of the library or the array differ in having anon-antibody protein sequence that comprises a portion of the ultralongCDR3. Such libraries or arrays may contain multiple non-antibody proteinsequences, including for chemokines, growth factors, peptides,cytokines, cell surface proteins, serum proteins, toxins, extracellularmatrix proteins, clotting factors, secreted proteins, viral or bacterialproteins, etc. The non-antibody protein sequence may be of human ornon-human origin and may be comprised of a portion of a non-antibodyprotein such as a peptide or domain. The non-antibody protein sequenceof the ultralong CDR3 may contain mutations from its natural sequence,including amino acid changes (e.g., substitutions), or insertions ordeletions. Engineering additional amino acids at the junction betweenthe non-antibody sequence within the ultralong CDR3 may be done tofacilitate or enhance proper folding of the non-antibody sequence withinthe antibody.

In another embodiment, the present disclosure provides libraries orarrays of antibodies comprising ultralong CDR3 sequences wherein atleast two members of the library or the array differ in having anon-bovine sequence. The non-bovine sequence can be derived from orbased on a non-bovine mammal sequence. For example, the non-bovinesequence can be derived from or based on a human, mouse, rat, sheep,dog, and/or goat sequence. The non-bovine sequence can be within theultralong CDR3. Alternatively, the non-bovine sequence is linked orattached to the ultralong CDR3 sequence. The non-bovine sequence can bederived from or based on at least a portion of an antibody sequence. Theantibody sequence can encode a variable region, constant region or acombination thereof.

In another embodiment, the present disclosure provides libraries orarrays of antibodies comprising ultralong CDR3 sequences wherein atleast two members of the library or array differ in having a cytoxicagent or therapeutic polypeptide that is conjugated to the ultralongCDR3. The cytoxic agent or therapeutic polypeptide may include, but isnot limited to, a chemotherapeutic agent, a drug, a growth inhibitoryagent, a toxin (e.g., an enzymatically active toxin of bacterial,fungal, plant, or animal origin, or fragments thereof), or a radioactiveisotope (e.g., a radioconjugate). The cytotoxic agent or therapeuticpolypeptide can be encoded by a non-antibody sequence.

In another embodiment, the present disclosure provides libraries orarrays of antibodies comprising ultralong CDR3 sequences wherein atleast two members of the library or array differ in binding to targets.The target can be a protein target. The protein target can be atransmembrane protein target. Such transmembrane targets may include,but are not limited to, GPCRs, ion channels, transporters, and cellsurface receptors.

The libraries or the arrays of the present disclosure may be in severalformats well known in the art. The library or the array may be anaddressable library or an addressable array. The library or array may bein display format, for example, the antibody sequences may be expressedon phage, ribosomes, mRNA, yeast, or mammalian cells.

Cells

The present disclosure provides cells comprising genetic sequencesencoding antibodies comprising ultralong CDR3 sequences or portionsthereof. The present disclosure provides cells comprising geneticsequences encoding antibodies comprising at least a portion of a knobdomain or at least a portion of a knob domain of an ultralong CDR3sequence.

The present disclosure provides cells comprising genetic sequences(e.g., genes, nucleic acids, polynucleotides) encoding an ultralong CDR3or portion thereof. The present disclosure also provides cellscomprising genetic sequences (e.g., genes, nucleic acids,polynucleotides) encoding the knob domain and/or knob domain of anultralong CDR3.

In an embodiment, the present disclosure provides cells expressing anantibody comprising an ultralong CDR3. The cells may be prokaryotic oreukaryotic, and an antibody comprising an ultralong CDR3 may beexpressed on the cell surface or secreted into the media. When displayedon the cell surface an antibody preferentially contains a motif forinsertion into the plasmid membrane such as a membrane spanning domainat the C-terminus or a lipid attachment site. For bacterial cells, anantibody comprising an ultralong CDR3 may be secreted into theperiplasm. When the cells are eukaryotic, they may be transientlytransfected with genetic sequences encoding an antibody comprising anultralong CDR3. Alternatively, a stable cell line or stable pools may becreated by transfecting or transducing genetic sequences encoding anantibody comprising an ultralong CDR3 by methods well known to those ofskill in the art. Cells can be selected by fluorescence activated cellsorting (FACS) or through selection for a gene encoding drug resistance.Cells useful for producing antibodies comprising ultralong CDR3sequences include prokaryotic cells like E. coli, eukaryotic cells likethe yeasts Saccharomyces cerevisiae and Pichia pastoris, insect cells(e.g., Sf9, Hi5), chinese hamster ovary (CHO) cells, monkey cells likeCOS-1, or human cells like HEK-293, HeLa, SP-1.

Library Methods

The present disclosure provides methods for making libraries comprisingantibodies comprising ultralong CDR3 sequences. Methods for makinglibraries of spatially addressed libraries are described in WO2010/054007. Methods of making libraries in yeast, phage, E. coli, ormammalian cells are well known in the art.

The present disclosure also provides methods of screening libraries ofantibodies comprising ultralong CDR3 sequences.

DEFINITIONS

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the exemplary embodiments (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein is merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the exemplary embodiments and doesnot pose a limitation on the scope of the exemplary embodimentsotherwise claimed. No language in the specification should be construedas indicating any non-claimed element essential to the practice of theexemplary embodiments.

An “ultralong CDR3” or an “ultralong CDR3 sequence”, usedinterchangeably herein, comprises a CDR3 or CDR3 sequence that is notderived from a human antibody sequence. An ultralong CDR3 may be 35amino acids in length or longer, for example, 40 amino acids in lengthor longer, 45 amino acids in length or longer, 50 amino acids in lengthor longer, 55 amino acids in length or longer, or 60 amino acids inlength or longer. The length of the ultralong CDR3 may include anon-antibody sequence. An ultralong CDR3 may comprise at least a portionof a knob domain and/or knob domain. An ultralong CDR3 may comprise anon-antibody sequence, including, for example, a cytokine, chemokine,growth factor or hormone sequence. Preferably, the ultralong CDR3 is aheavy chain CDR3 (CDR-H3 or CDRH3). Preferably, the ultralong CDR3 is asequence derived from or based on a ruminant (e.g., bovine) sequence. Anultralong CDR3 may comprise at least 3 or more cysteine residues, forexample, 4 or more cysteine residues, 6 or more cysteine residues, or 8or more cysteine residues. An ultralong CDR3 may comprise one or morecysteine motifs. An ultralong CDR3 may comprise an amino acid sequencethat is derived from or based on SEQ ID NOS: 23-44 or is encoded by aDNA sequence that is derived from or based on SEQ ID NOS: 2-22. Avariable region that comprises an ultralong CDR3 may include an aminoacid sequence that is derived from or based on SEQ ID NOS: 23-44 or isencoded by a DNA sequence that is derived from or based on SEQ ID NOS:2-22. Such a sequence may be derived from or based on a bovine germlineVH gene sequence. An ultralong CDR3 may comprise a sequence derived fromor based on a non-human DH gene sequence (see, e.g., Koti, et al. (2010)Mol. Immunol. 47: 2119-2128). An ultralong CDR3 may comprise a sequencederived from or based on a JH sequence, (see e.g., Hosseini, et al.(2004) Int. Immunol. 16: 843-852). In an embodiment, an ultralong CDR3may comprise a sequence derived from or based on a non-human VH sequenceand/or a sequence derived from or based on a non-human DH sequenceand/or a sequence derived from or based on a JH sequence, and optionallyan additional sequence comprising two to six amino acids or more suchas, for example, between the VH derived sequence and the DH derivedsequence. In another embodiment, an ultralong CDR3 may comprise asequence that is about 50% or more homologous to a sequence derived fromor based on SEQ ID NOS: 23-44. For example, the ultralong CDR3 maycomprise a sequence that is about 60%, 70%, 80%, 85%, 90%, 95%, 97% ormore homologous to a sequence derived from or based on SEQ ID NOS:23-44. In another embodiment, an ultralong CDR3 may comprise a sequencethat aligns to 5 or more amino acids to a sequence derived from or basedSEQ ID NOS: 23-44. For example, the ultralong CDR3 may comprise asequence that aligns to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 ormore amino acids to a sequence derived from or based SEQ ID NOS: 23-44.In another embodiment, an ultralong CDR3 may comprise a sequence thatcomprises 5 or more consecutive amino acids to a sequence derived fromor based SEQ ID NOS: 23-44. For example, the ultralong CDR3 may comprisea sequence that comprises 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60or more consecutive amino acids to a sequence derived from or based SEQID NOS: 23-44. In another embodiment, an ultralong CDR3 may comprise asequence that is about 50% or more homologous to a DNA sequence that isderived from or based on a SEQ ID NOS: 2-22. For example, the ultralongCDR3 may comprise a sequence that is about 60%, 70%, 80%, 85%, 90%, 95%,97% or more homologous to a DNA sequence that is derived from or basedon SEQ ID NOS: 2-22. In another embodiment, an ultralong CDR3 maycomprise a sequence that aligns to 5 or more nucleic acids to a DNAsequence that is derived from or based SEQ ID NOS: 2-22. For example,the ultralong CDR3 may comprise a sequence that aligns to 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 80, 100, 120, 140, 150, 175, 200, 225,250, 275, 300, 350, 400, 450, 500, 550, 600 or more nucleic acids to aDNA sequence that is derived from or based SEQ ID NOS: 2-22. In anotherembodiment, an ultralong CDR3 may comprise a sequence that comprises 5or more consecutive nucleic acids to a DNA sequence that is derived fromor based SEQ ID NOS: 2-22. For example, the ultralong CDR3 may comprisea sequence that comprises 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,80, 100, 120, 140, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450,500, 550, 600 or more consecutive amino acids to a DNA sequence that isderived from or based SEQ ID NOS: 2-22. In another embodiment, anultralong CDR3 may comprise a sequence that is about 50% or morehomologous to a sequence derived from or based on a knob domainsequence. For example, the ultralong CDR3 may comprise a sequence thatis about 60%, 70%, 80%, 85%, 90%, 95%, 97% or more homologous to asequence derived from or based on a knob domain sequence. In anotherembodiment, an ultralong CDR3 may comprise a sequence that aligns to 5or more amino acids to a sequence derived from or based a knob domainsequence. For example, the ultralong CDR3 may comprise a sequence thataligns to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more aminoacids to a sequence derived from or based a knob domain sequence Inanother embodiment, an ultralong CDR3 may comprise a sequence thatcomprises 5 or more consecutive amino acids to a sequence derived fromor based a knob domain sequence For example, the ultralong CDR3 maycomprise a sequence that comprises 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60 or more consecutive amino acids to a sequence derived from orbased a knob domain sequence. In another embodiment, an ultralong CDR3may comprise a sequence that is about 50% or more homologous to asequence derived from or based on a knob domain sequence. For example,the ultralong CDR3 may comprise a sequence that is about 60%, 70%, 80%,85%, 90%, 95%, 97% or more homologous to a sequence derived from orbased on a knob domain sequence. In another embodiment, an ultralongCDR3 may comprise a sequence that aligns to 5 or more amino acids to asequence derived from or based a knob domain sequence For example, theultralong CDR3 may comprise a sequence that aligns to 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60 or more amino acids to a sequence derivedfrom or based a knob domain sequence In another embodiment, an ultralongCDR3 may comprise a sequence that comprises 5 or more consecutive aminoacids to a sequence derived from or based a stalk domain sequence Forexample, the ultralong CDR3 may comprise a sequence that comprises 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more consecutive aminoacids to a sequence derived from or based a stalk domain sequence. Theantibodies disclosed herein may comprise at least a portion of anultralong CDR3 derived from or based on a sequence of any of theultralong CDR3s disclosed herein. The sequence of the ultralong CDR3 ora portion thereof may be modified or altered to contain one or morenon-bovine antibody-based nucleotides and/or amino acids. Themodifications and/or alterations in the sequence of the ultralong CDR3or portion thereof may improve one or more features of the expressedantibody. For example, the modifications and/or alterations may improveexpression, folding, half-life, activity and/or solubility of theantibody.

An “isolated” biological molecule, such as the various polypeptides,polynucleotides, and antibodies disclosed herein, refers to a biologicalmolecule that has been identified and separated and/or recovered from atleast one component of its natural environment.

“Antagonist” refers to any molecule that partially or fully blocks,inhibits, or neutralizes an activity (e.g., biological activity) of apolypeptide. Also encompassed by “antagonist” are molecules that fullyor partially inhibit the transcription or translation of mRNA encodingthe polypeptide. Suitable antagonist molecules include, e.g., antagonistantibodies or antibody fragments; fragments or amino acid sequencevariants of a native polypeptide; peptides; antisense oligonucleotides;small organic molecules; and nucleic acids that encode polypeptideantagonists or antagonist antibodies. Reference to “an” antagonistencompasses a single antagonist or a combination of two or moredifferent antagonists.

“Agonist” refers to any molecule that partially or fully mimics abiological activity of a polypeptide. Also encompassed by “agonist” aremolecules that stimulate the transcription or translation of mRNAencoding the polypeptide. Suitable agonist molecules include, e.g.,agonist antibodies or antibody fragments; a native polypeptide;fragments or amino acid sequence variants of a native polypeptide;peptides; antisense oligonucleotides; small organic molecules; andnucleic acids that encode polypeptides agonists or antibodies. Referenceto “an” agonist encompasses a single agonist or a combination of two ormore different agonists.

An “isolated” antibody refers to one which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody (e.g., asdetermined by the Lowry method), and preferably to more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence (e.g., by use of a spinningcup sequenator), or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions (e.g., using Coomassie™ blue or, preferably,silver stain). Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Similarly, isolated antibody includesthe antibody in medium around recombinant cells. An isolated antibodymay be prepared by at least one purification step.

An “isolated” nucleic acid molecule refers to a nucleic acid moleculethat is identified and separated from at least one contaminant nucleicacid molecule with which it is ordinarily associated in the naturalsource of the antibody nucleic acid. An isolated nucleic acid moleculeis other than in the form or setting in which it is found in nature.Isolated nucleic acid molecules therefore are distinguished from thenucleic acid molecule as it exists in natural cells. However, anisolated nucleic acid molecule includes a nucleic acid moleculecontained in cells that express an antibody where, for example, thenucleic acid molecule is in a chromosomal location different from thatof natural cells.

Variable domain residue numbering as in Kabat or amino acid positionnumbering as in Kabat, and variations thereof, refers to the numberingsystem used for heavy chain variable domains or light chain variabledomains of the compilation of antibodies in Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (e.g.,residue 52a according to Kabat) after residue 52 of H2 and insertedresidues (e.g., residues 82a, 82b, and 82c, etc according to Kabat)after heavy chain FR residue 82. The Kabat numbering of residues may bedetermined for a given antibody by alignment at regions of homology ofthe sequence of the antibody with a “standard” Kabat numbered sequence.

“Substantially similar,” or “substantially the same”, refers to asufficiently high degree of similarity between two numeric values(generally one associated with an antibody disclosed herein and theother associated with a reference/comparator antibody) such that one ofskill in the art would consider the difference between the two values tobe of little or no biological and/or statistical significance within thecontext of the biological characteristic measured by said values (e.g.,Kd values). The difference between said two values is preferably lessthan about 50%, preferably less than about 40%, preferably less thanabout 30%, preferably less than about 20%, preferably less than about10% as a function of the value for the reference/comparator antibody.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, “binding affinity” refers to intrinsic bindingaffinity which reflects a 1:1 interaction between members of a bindingpair (e.g., antibody and antigen). The affinity of a molecule X for itspartner Y can generally be represented by the dissociation constant.Affinity can be measured by common methods known in the art, includingthose described herein. Low-affinity antibodies generally bind antigenslowly and tend to dissociate readily, whereas high-affinity antibodiesgenerally bind antigen faster and tend to remain bound longer. A varietyof methods of measuring binding affinity are known in the art, any ofwhich can be used for purposes of the present disclosure.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”can be determined with a surface plasmon resonance technique such asBiacore (e.g., Biacore A100, Biacore™-2000, Biacore™-3000, Biacore,Inc., Piscataway, N.J.) carboxymethylated dextran biosensor chips (CM5,Biacore Inc.) and according to the supplier's instructions.

“Vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments may be ligated. Another type of vector isa phage vector. Another type of vector is a viral vector, whereinadditional DNA segments may be ligated into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors having a bacterial originof replication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “recombinant vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. Accordingly, “plasmid” and “vector” may, at times,be used interchangeably as the plasmid is a commonly used form ofvector.

“Gene” refers to a nucleic acid (e.g., DNA) sequence that comprisescoding sequences necessary for the production of a polypeptide,precursor, or RNA (e.g., mRNA, rRNA, tRNA). The polypeptide can beencoded by a full length coding sequence or by any portion of the codingsequence so long as the desired activity or functional properties (e.g.,enzymatic activity, ligand binding, signal transduction, immunogenicity,etc.) of the full-length or fragment are retained. The term alsoencompasses the coding region of a structural gene and the sequenceslocated adjacent to the coding region on both the 5′ and 3′ ends for adistance of about 1 kb or more on either end such that the genecorresponds to the length of the full-length mRNA. Sequences located 5′of the coding region and present on the mRNA are referred to as 5′non-translated sequences. Sequences located 3′ or downstream of thecoding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns cancontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide. In addition to containing introns, genomic forms ofa gene can also include sequences located on both the 5′ and 3′ end ofthe sequences that are present on the RNA transcript. These sequencesare referred to as “flanking” sequences or regions (these flankingsequences are located 5′ or 3′ to the non-translated sequences presenton the mRNA transcript). The 5′ flanking region can contain regulatorysequences such as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region can contain sequencesthat direct the termination of transcription, post transcriptionalcleavage and polyadenylation.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refers to polymers of nucleotides of any length, and include DNA andRNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase, or bya synthetic reaction. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and their analogs. Ifpresent, modification to the nucleotide structure may be imparted beforeor after assembly of the polymer. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after synthesis, such as by conjugation with a label.Other types of modifications include, for example, “caps”, substitutionof one or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid or semi-solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and a basic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages may be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S(“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

“Oligonucleotide” refers to short, generally single stranded, generallysynthetic polynucleotides that are generally, but not necessarily, lessthan about 200 nucleotides in length. The terms “oligonucleotide” and“polynucleotide” are not mutually exclusive. The description above forpolynucleotides is equally and fully applicable to oligonucleotides.

“Stringent hybridization conditions” refer to conditions under which aprobe will hybridize to its target subsequence, typically in a complexmixture of nucleic acids, but to no other sequences. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

“Recombinant” when used with reference to a cell, nucleic acid, protein,antibody or vector indicates that the cell, nucleic acid, protein orvector has been modified by the introduction of a heterologous nucleicacid or protein, the alteration of a native nucleic acid or protein, orthat the cell is derived from a cell so modified. For example,recombinant cells express genes that are not found within the native(non-recombinant) form of the cell or express native genes that areoverexpressed or otherwise abnormally expressed such as, for example,expressed as non-naturally occurring fragments or splice variants. Bythe term “recombinant nucleic acid” herein is meant nucleic acid,originally formed in vitro, in general, by the manipulation of nucleicacid, e.g., using polymerases and endonucleases, in a form not normallyfound in nature. In this manner, operably linkage of different sequencesis achieved. Thus an isolated nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis disclosure. It is understood that once a recombinant nucleic acidis made and introduced into a host cell or organism, it will replicatenon-recombinantly, e.g., using the in vivo cellular machinery of thehost cell rather than in vitro manipulations; however, such nucleicacids, once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposesdisclosed herein. Similarly, a “recombinant protein” is a protein madeusing recombinant techniques, e.g., through the expression of arecombinant nucleic acid as depicted herein.

“Percent (%) amino acid sequence identity” with respect to a peptide orpolypeptide sequence refers to the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegAlign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared.

“Polypeptide,” “peptide,” “protein,” and “protein fragment” may be usedinterchangeably to refer to a polymer of amino acid residues. The termsapply to amino acid polymers in which one or more amino acid residue isan artificial chemical mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers andnon-naturally occurring amino acid polymers.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that functionsimilarly to the naturally occurring amino acids. Naturally occurringamino acids are those encoded by the genetic code, as well as thoseamino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refersto compounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an alpha carbon that is bound to a hydrogen,a carboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs can have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. “Amino acid variants” refers to amino acidsequences. With respect to particular nucleic acid sequences,conservatively modified variants refers to those nucleic acids whichencode identical or essentially identical amino acid sequences, or wherethe nucleic acid does not encode an amino acid sequence, to essentiallyidentical or associated (e.g., naturally contiguous) sequences. Becauseof the degeneracy of the genetic code, a large number of functionallyidentical nucleic acids encode most proteins. For instance, the codonsGCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at everyposition where an alanine is specified by a codon, the codon can bealtered to another of the corresponding codons described withoutaltering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes silent variations of the nucleic acid. One ofskill will recognize that in certain contexts each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, silentvariations of a nucleic acid which encodes a polypeptide is implicit ina described sequence with respect to the expression product, but notwith respect to actual probe sequences. As to amino acid sequences, oneof skill will recognize that individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide, or protein sequencewhich alters, adds or deletes a single amino acid or a small percentageof amino acids in the encoded sequence is a “conservatively modifiedvariant” including where the alteration results in the substitution ofan amino acid with a chemically similar amino acid. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. Such conservatively modified variants are in additionto and do not exclude polymorphic variants, interspecies homologs, andalleles disclosed herein. Typically conservative substitutionsinclude: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingsimilar structural characteristics. While antibodies may exhibit bindingspecificity to a specific antigen, immunoglobulins may include bothantibodies and other antibody-like molecules which generally lackantigen specificity. Polypeptides of the latter kind are, for example,produced at low levels by the lymph system and at increased levels bymyelomas.

“Antibody”, “immunoglobulin” and “immunoglobulin construct” are usedinterchangeably in the broadest sense and include monoclonal antibodies(e.g., full length or intact monoclonal antibodies), polyclonalantibodies, multivalent antibodies, multispecific antibodies (e.g.,bispecific antibodies so long as they exhibit the desired biologicalactivity) and may also include certain antibody fragments (as describedin greater detail herein). The term “antibody” can refer to a fulllength antibody or a portion thereof. An antibody can refer to a peptidecomprising at least one antibody sequence. The antibody sequence cancomprise 5 or more amino acids of an antibody sequence. For example theantibody sequence can comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more amino acids of an antibody sequence. The 5 ormore amino acids may be consecutive amino acids of an antibody sequence.Alternatively, the 5 or more amino acids are non-consecutive amino acidsof an antibody sequence. For example, the 5 or more amino acids maycomprise a conserved motif within the antibody sequence. For example,the 5 or more amino acids may comprise a conserved motif within anultralong CDR3 sequence. An antibody can be human, humanized, fullyhuman and/or affinity matured. An antibody can be a chimeric antibody.An antibody can be a recombinant, engineered, or synthetic antibody. Anantibody may be a bovine, bovine engineered, fully bovine and/oraffinity matured. The bovine engineered antibody may comprise one ormore nucleotides or peptides derived from a bovine antibody sequence. Afully bovine antibody may comprise replacing one or more nucleotides orpeptides from a non-bovine antibody sequence with one or morenucleotides or peptides based on a bovine antibody sequence. An antibodymay refer to immunoglobulins and immunoglobulin portions, whethernatural or partially or wholly synthetic, such as recombinantlyproduced, including any portion thereof containing at least a portion ofthe variable region of the immunoglobulin molecule that is sufficient toform an antigen binding site. Hence, an antibody or portion thereofincludes any protein having a binding domain that is homologous orsubstantially homologous to an immunoglobulin antigen binding site. Forexample, an antibody may refer to an antibody that contains two heavychains (which can be denoted H and H′) and two light chains (which canbe denoted L and L′), where each heavy chain can be a full-lengthimmunoglobulin heavy chain or a portion thereof sufficient to form anantigen binding site (e.g. heavy chains include, but are not limited to,VH, chains VH-CH1 chains and VH-CH1-CH2-CH3 chains), and each lightchain can be a full-length light chain or a thereof sufficient to forman antigen binding site (e.g. light chains include, but are not limitedto, VL chains and VL-CL chains). Each heavy chain (H and H′) pairs withone light chain (L and L′, respectively). Typically, antibodiesminimally include all or at least a portion of the variable heavy (VH)chain and/or the variable light (VL) chain. The antibody also caninclude all or a portion of the constant region. For example, afull-length antibody is an antibody having two full-length heavy chains(e.g. VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length lightchains (VL-CL) and hinge regions, such as antibodies produced byantibody secreting B cells and antibodies with the same domains that areproduced synthetically. Additionally, an “antibody” refers to a proteinof the immunoglobulin family or a polypeptide comprising fragments of animmunoglobulin that is capable of noncovalently, reversibly, and in aspecific manner binding a corresponding antigen. An exemplary antibodystructural unit comprises a tetramer. Each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one “light”(about 25 kD) and one “heavy” chain (about 50-70 kD), connected througha disulfide bond. The recognized immunoglobulin genes include the κ, λ,α, γ, δ, ε, and μ constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE,respectively. The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. The terms variable light chain (VL) and variable heavychain (VH) refer to these regions of light and heavy chainsrespectively. In some instances, the antibodies provided herein compriseat least one immunoglobulin domain from an avian antibody, reptilianantibody, amphibian antibody, insect antibody, or chimeric combinationsthereof. The antibodies can comprise at least one immunoglobulin domainfrom a chimeric antibody. The chimeric antibody can be derived from twoor more different species (e.g., mouse and human, bovine and human). Theantibodies can comprise at least one immunoglobulin domain from anengineered, recombinant or synthetic antibody. In some instances,engineered, recombinant or synthetic antibodies are created usingantibody genes made in a laboratory or taken from cells. The antibodygenes can be derived from one or more mammals. For example, the antibodygenes are derived from a human. The antibody genes may be derived from abovine. Alternatively, or additionally, the antibodies disclosed hereincomprise at least one immunoglobulin domain from a humanized, humanengineered or fully human antibody. The antibody may comprise antibodysequences from two or more different antibodies. The two or moredifferent antibodies may be from the same species. For example, thespecie may be a bovine specie, human specie, or murine specie. The twoor more different antibodies may be from the same type of animal. Forexample the two or more different antibodies may be from a cow. The twoor more different antibodies may be from a human. Alternatively, the twoor more different antibodies are from different species. For example,the two or more different antibodies are from a human specie and bovinespecie. In another example, the two or more diffent antibodies are froma bovine specie and a non-bovine specie. In another example, the two ormore different antibodies are from a human specie and a non-humanspecie. The two or more different antibodies may be from differentanimals. For example, the two different animals are a human and a cow.The different animals may be from the same specie. For example, thedifferent animals may be a cow and a water buffalo.

“Variable” refers to the fact that certain portions of the variabledomains (also referred to as variable regions) differ extensively insequence among antibodies and are used in the binding and specificity ofeach particular antibody for its particular antigen. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments calledcomplementarity-determining regions (CDRs) or hypervariable regions(HVRs) both in the light-chain and the heavy-chain variable domains.CDRs include those specified as Kabat, Chothia, and IMGT as shown hereinwithin the variable region sequences. The more highly conserved portionsof variable domains are called the framework (FR). The variable domainsof native heavy and light chains each comprise four FR regions, largelyadopting a β-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen-binding site of antibodies (see Kabat etal., Sequences of Proteins of Immunological Interest, Fifth Edition,National Institute of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” refers to an antibody fragment which contains anantigen-recognition and antigen-binding site. In a two-chain Fv species,this region consists of a dimer of one heavy and one light chainvariable domain in non-covalent association. In a single chain Fv (scFv)species, one heavy chain and one light chain variable domain can becovalently linked by a flexible peptide linker such that the light andheavy chains can associate in a “dimeric” structure analogous to that ina two-chain Fv (scFv) species. It is in this configuration that thethree CDRs of each variable domain interact to define an antigen-bindingsite on the surface of the VH-VL dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CHI) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, single-chain Fvs (scFv), Fv, dsFv, diabody (e.g., (ds Fv)₂), Fdand Fd′ fragments Fab fragments, Fd fragments, scFv fragments, linearantibodies, single-chain antibody molecules, minibodies, flexminibodies, bispecific fragments, and multispecific antibodies formedfrom antibody fragments (see, for example, Methods in Molecular Biology,Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003); Chapter 1; p 3-25, Kipriyanov). Other known fragments include,but are not limited to, scFab fragments (Hust et al., BMC Biotechnology(2007), 7:14). In one embodiment, an antibody fragment comprises anantigen binding site of the intact antibody and thus retains the abilityto bind antigen. In another embodiment, an antibody fragment, forexample one that comprises the Fc region, retains at least one of thebiological functions normally associated with the Fc region when presentin an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise on antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment. Foranother example, an antibody fragment or antibody portion refers to anyportion of a full-length antibody that is less than full length butcontains at least a portion of the variable region of the antibodysufficient to form an antigen binding site (e.g. one or more CDRs) andthus retains the a binding specificity and/or an activity of thefull-length antibody; antibody fragments include antibody derivativesproduced by enzymatic treatment of full-length antibodies, as well assynthetically, e.g. recombinantly produced derivatives.

A “dsFv” refers to an Fv with an engineered intermolecular disulfidebond, which stabilizes the VH-VL pair.

A “Fd fragment” refers to a fragment of an antibody containing avariable domain (VH) and one constant region domain (CH1) of an antibodyheavy chain.

A “Fab fragment” refers to an antibody fragment that contains theportion of the full-length antibody that would results from digestion ofa full-length immunoglobulin with papain, or a fragment having the samestructure that is produced synthetically, e.g. recombinantly. A Fabfragment contains a light chain (containing a VL and CL portion) andanother chain containing a variable domain of a heavy chain (VH) and oneconstant region domain portion of the heavy chain (CH1); it can berecombinantly produced.

A “F(ab′)2 fragment” refers to an antibody fragment that results fromdigestion of an immunoglobulin with pepsin at pH 4.0-4.5, or asynthetically, e.g. recombinantly, produced antibody having the samestructure. The F(ab′)2 fragment contains two Fab fragments but whereeach heavy chain portion contains an additional few amino acids,including cysteine residues that form disulfide linkages joining the twofragments; it can be recombinantly produced.

A “Fab′ fragment” refers to a fragment containing one half (one heavychain and one light chain) of the F(ab′)2 fragment.

A “Fd′ fragment refers to a fragment of an antibody containing one heavychain portion of a F(ab′)2 fragment.

A “Fv′ fragment” refers to a fragment containing only the VH and VLdomains of an antibody molecule.

A “scFv fragment” refers to an antibody fragment that contains avariable light chain (VL) and variable heavy chain (VH), covalentlyconnected by a polypeptide linker in any order. The linker is of alength such that the two variable domains are bridged withoutsubstantial interference. Exemplary linkers are (Gly-Ser)n residues withsome Glu or Lys residues dispersed throughout to increase solubility.

Diabodies are dimeric scFv; diabodies typically have shorter peptidelinkers than scFvs, and they preferentially dimerize.

“HsFv” refers to antibody fragments in which the constant domainsnormally present in a Fab fragment have been substituted with aheterodimeric coiled-coil domain (see, e.g., Arndt et al. (2001) J Mol.Biol. 7:312:221-228).

“Hypervariable region”, “HVR”, or “HV”, as well as “complementarydetermining region” or “CDR”, may refer to the regions of an antibodyvariable domain which are hypervariable in sequence and/or formstructurally defined loops. Generally, antibodies comprise sixhypervariable or CDR regions; three in the VH (H1, H2, H3), and three inthe VL (L1, L2, L3). A number of hypervariable region or CDRdelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (Kabat CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops,(Chothia “CDRs”) and are used by Oxford Molecular's AbM antibodymodeling software. The “contact” hypervariable regions are based on ananalysis of the available complex crystal structures. The residues fromeach of these hypervariable regions are noted below. (See also, forexample, FIG. 1 and bold, italicized text for Kabat CDRs and underlinedtext for Chothia CDRs for 12.3 ICI antibody).

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

IMGT referes to the international ImMunoGeneTics Information System, asdescribed by Lefrace et al., Nucl. Acids, Res. 37; D1006-D1012 (2009),including for example, IMGT designated CDRs for antibodies (see also,for example, FIG. 1 and bracketed text for 12.3 1C1 antibody).

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3)in the VH. The variable domain residues are numbered according to Kabatet al., Supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined. “Frameworkregions” (FRs) are the domains within the antibody variable regiondomains comprising framework residues that are located within the betasheets; the FR regions are comparatively more conserved, in terms oftheir amino acid sequences, than the hypervariable regions.

“Monoclonal antibody” refers to an antibody from a population ofsubstantially homogeneous antibodies, that is, for example, theindividual antibodies comprising the population are identical and/orbind the same epitope(s), except for possible variants that may ariseduring production of the monoclonal antibody, such variants generallybeing present in minor amounts. Such monoclonal antibody typicallyincludes an antibody comprising a polypeptide sequence that binds atarget, wherein the target-binding polypeptide sequence was obtained bya process that includes the selection of a single target bindingpolypeptide sequence from a plurality of polypeptide sequences. Forexample, the selection process can be the selection of a unique clonefrom a plurality of clones, such as a pool of hybridoma clones, phageclones or recombinant DNA clones. It should be understood that theselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this disclosure. In contrast to polyclonalantibody preparations which typically include different antibodiesdirected against different determinants (e.g., epitopes), eachmonoclonal antibody of a monoclonal antibody preparation is directedagainst a single determinant on an antigen. In addition to theirspecificity, the monoclonal antibody preparations are advantageous inthat they are typically uncontaminated by other immunoglobulins. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present disclosure may be made by a variety oftechniques, including, for example, the hybridoma method (e.g., Kohleret al., Nature, 256:495 (1975); Harlow et al., Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerlinget al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681,(Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat.No. 4,816,567), phage display technologies (see, e.g., Clackson et al.,Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597(1991); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al.,J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci.USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods284(1-2):119-132 (2004), and technologies for producing human orhuman-like antibodies in animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences(see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741;Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Yearin Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806; 5,569,825; 5,591,669;5,545,807; WO 1997/17852; U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al.,Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859(1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., NatureBiotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93(1995)).

“Humanized” or “Human engineered” forms of non-human (e.g., murine,bovine) antibodies are chimeric antibodies that contain amino acidsrepresented in human immunoglobulin sequences, including, for example,wherein minimal sequence is derived from non-human immunoglobulin. Forexample, humanized antibodies may be human antibodies in which somehypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in non-human (e.g., rodent)antibodies. Alternatively, humanized or human engineered antibodies maybe non-human (e.g., rodent) antibodies in which some residues aresubstituted by residues from analogious sites in human antibodies (see,e.g., U.S. Pat. No. 5,766,886). Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody, including, for example non-antibody sequences such as achemokine, growth factor, peptide, cytokine, cell surface protein, serumprotein, toxin, extracellular matrix protein, clotting factor, orsecreted protein sequence. These modifications may be made to furtherrefine antibody performance. Humanized antibodies include humanengineered antibodies, for example, as described by U.S. Pat. No.5,766,886, including methods for preparing modified antibody variabledomains. A humanized antibody may comprise substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the hypervariable loops correspond to those of a non-humanimmunoglobulin and all or substantially all of the FRs are those of ahuman immunoglobulin sequence. A humanized antibody optionally may alsocomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See alsothe following review articles and references cited therein: Vaswani andHamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris,Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr.Op. Biotech. 5:428-433 (1994).

“Hybrid antibodies” refer to immunoglobulin molecules in which pairs ofheavy and light chains from antibodies with different antigenicdeterminant regions are assembled together so that two differentepitopes or two different antigens can be recognized and bound by theresulting tetramer.

“Chimeric” antibodies (immunoglobulins) have a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (see e.g., Morrison et al.,Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Humanized antibodyrefers to a subset of chimeric antibodies.

“Single-chain Fv” or “scFv” antibody fragments may comprise the VH andVL domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994).

An “antigen” refers to a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Preferably, the target antigen is a polypeptide.

“Epitope” or “antigenic determinant”, used interchangeably herein, referto that portion of an antigen capable of being recognized andspecifically bound by a particular antibody. When the antigen is apolypeptide, epitopes can be formed both from contiguous amino acids andnoncontiguous amino acids juxtaposed by tertiary folding of a protein.Epitopes formed from contiguous amino acids are typically retained uponprotein denaturing, whereas epitopes formed by tertiary folding aretypically lost upon protein denaturing. An epitope typically includes atleast 3, and more usually, at least 5 or 8-10 amino acids in a uniquespatial conformation. Antibodies may bind to the same or a differentepitope on an antigen. Antibodies may be characterized in differentepitope bins. Whether an antibody binds to the same or different epitopeas another antibody (e.g., a reference antibody or benchmark antibody)may be determined by competition between antibodies in assays (e.g.,competitive binding assays).

Competition between antibodies may be determined by an assay in whichthe immunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen. Numerous types of competitive bindingassays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay or enzyme-linked immunosorbent assay (EIA or ELISA),sandwich competition assay including an ELISA assay (see Stahli et al.,Methods in Enzymology 9:242-253 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, “Antibodies, A Laboratory Manual,”Cold Spring Harbor Press (1988)); solid phase direct label RIA using1-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.,32:77-82 (1990)). Competition binding assays may be performed usingSurface Plasmon Resonance (SPR), for example, with a Biacore® instrumentfor kinetic analysis of binding interactions. In such an assay, anantibody comprising an ultralong CDR3 of unknown epitope specificity maybe evaluated for its ability to compete for binding against a comparatorantibody (e.g., a BA1 or BA2 antibody as described herein). An assay mayinvolve the use of purified antigen bound to a solid surface or cellsbearing either of these, an unlabeled test immunoglobulin and a labeledreference immunoglobulin. Competitive inhibition may be measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. An assay (competing antibodies) may includeantibodies binding to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least50%, or at least about 70%, or at least about 80%, or least about 90%,or at least about 95%, or at least about 99% or about 100% for acompetitor antibody.

That an antibody “selectively binds” or “specifically binds” means thatthe antibody reacts or associates more frequently, more rapidly, withgreater duration, with greater affinity, or with some combination of theabove to an antigen or an epitope than with alternative substances,including unrelated proteins. “Selectively binds” or “specificallybinds” may mean, for example, that an antibody binds to a protein with aK_(D) of at least about 0.1 mM, or at least about 1 μM or at least about0.1 μM or better, or at least about 0.01 μM or better. Because of thesequence identity between homologous proteins in different species,specific binding can include an antibody that recognizes a given antigenin more than one species.

“Non-specific binding” and “background binding” when used in referenceto the interaction of an antibody and a protein or peptide refer to aninteraction that is not dependent on the presence of a particularstructure (e.g., the antibody is binding to proteins in general ratherthat a particular structure such as an epitope).

“Diabodies” refer to small antibody fragments with two antigen-bindingsites, which fragments comprise a heavy-chain variable domain (VH)connected to a light-chain variable domain (VL) in the same polypeptidechain (VH-VL). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, forexample, EP 404,097; WO 93/11161; and Hollinger et. al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “human antibody” refers to one which possesses an amino acid sequencewhich corresponds to that of an antibody produced by a human and/or hasbeen made using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody refers to one with one or morealterations in one or more CDRs thereof which result in an improvementin the affinity of the antibody for antigen, compared to a parentantibody which does not possess those alteration(s). Preferred affinitymatured antibodies will have nanomolar or even picomolar affinities forthe target antigen. Affinity matured antibodies are produced byprocedures known in the art. Marks et al., Bio/Technology 10:779-783(1992) describes affinity maturation by VH and VL domain shuffling.Random mutagenesis of CDR and/or framework residues is described by:Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier etal., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004(1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins etal., J. Mol. Biol. 226:889-896 (1992).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: Clq bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, may be performed. Useful effectorcells for such assays include peripheral blood mononuclear cells (PBMC)and Natural Killer (NK) cells. Alternatively, or additionally, ADCCactivity of the molecule of interest may be assessed in vivo, e.g., in aanimal model such as that disclosed in Clynes et al. Proc. Natl. Acad.Sci. USA 95:652-656 (1998).

“Effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g., from blood.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaeron, Annu Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu Rev. Immuno19:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)) and regulates homeostasis ofimmunoglobulins. For example, antibody variants with improved ordiminished binding to FcRs have been described (see, e.g., Shields etal. J. Biol. Chem. 9(2): 6591-6604 (2001)).

Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997,Hinton 2004). Binding to human FcRn in vivo and serum half life of humanFcRn high affinity binding polypeptides can be assayed, e.g., intransgenic mice or transfected human cell lines expressing human FcRn,or in primates administered with the Fc variant polypeptides.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (Clq) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, for example, as described in Gazzano-Santoro etal., J. Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased Clq binding capability have been described (e.g.,see, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000)).

“Fc region-comprising polypeptide” refers to a polypeptide, such as anantibody or immunoadhesin (see definitions below), which comprises an Fcregion. The C-terminal lysine (residue 447 according to the EU numberingsystem) of the Fc region may be removed, for example, duringpurification of the polypeptide or by recombinant engineering thenucleic acid encoding the polypeptide.

“Blocking” antibody or an “antagonist” antibody refers to one whichinhibits or reduces biological activity of the antigen it binds.Preferred blocking antibodies or antagonist antibodies substantially orcompletely inhibit the biological activity of the antigen.

“Agonist” antibody refers to an antibody which mimics (e.g., partiallyor fully) at least one of the functional activities of a polypeptide ofinterest.

“Acceptor human framework” refers to a framework comprising the aminoacid sequence of a VL or VH framework derived from a humanimmunoglobulin framework, or from a human consensus framework. Anacceptor human framework “derived from” a human immunoglobulin frameworkor human consensus framework may comprise the same amino acid sequencethereof, or may contain pre-existing amino acid sequence changes. Wherepre-existing amino acid changes are present, preferably no more than 5and preferably 4 or less, or 3 or less, pre-existing amino acid changesare present.

A “human consensus framework” refers to a framework which represents themost commonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

“Disorder” or “disease” refers to any condition that would benefit fromtreatment with a substance/molecule (e.g., an antibody comprising anultralong CDR3 as disclosed herein) or method disclosed herein. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion.

“Treatment” refers to clinical intervention in an attempt to alter thenatural course of the individual or cell being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies disclosed herein are used to delaydevelopment of a disease or disorder.

“Individual” (e.g., a “subject”) refers to a vertebrate, preferably amammal, more preferably a human. Mammals include, but are not limitedto, farm animals (such as cows), sport animals, pets (such as cats, dogsand horses), primates, mice and rats.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, rodents (e.g., mice and rats), and monkeys;domestic and farm animals; and zoo, sports, laboratory, or pet animals,such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Insome embodiments, the mammal is selected from a human, rodent, ormonkey.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, including humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound thatis pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound.

“Pharmaceutically acceptable excipient, carrier or adjuvant” refers toan excipient, carrier or adjuvant that can be administered to a subject,together with at least one antibody of the present disclosure, and whichdoes not destroy the pharmacological activity thereof and is nontoxicwhen administered in doses sufficient to deliver a therapeutic amount ofthe compound.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient, or carrier with which at least one antibody of the presentdisclosure is administered.

“Providing a prognosis”, “prognostic information”, or “predictiveinformation” refer to providing information, including for example thepresence of cancer cells in a subject's tumor, regarding the impact ofthe presence of cancer (e.g., as determined by the diagnostic methods ofthe present disclosure) on a subject's future health (e.g., expectedmorbidity or mortality, the likelihood of getting cancer, and the riskof metastasis).

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented.

“Providing a diagnosis” or “diagnostic information” refers to anyinformation, including for example the presence of cancer cells, that isuseful in determining whether a patient has a disease or conditionand/or in classifying the disease or condition into a phenotypiccategory or any category having significance with regards to theprognosis of or likely response to treatment (either treatment ingeneral or any particular treatment) of the disease or condition.Similarly, diagnosis refers to providing any type of diagnosticinformation, including, but not limited to, whether a subject is likelyto have a condition (such as a tumor), whether a subject's tumorcomprises cancer stem cells, information related to the nature orclassification of a tumor as for example a high risk tumor or a low risktumor, information related to prognosis and/or information useful inselecting an appropriate treatment. Selection of treatment can includethe choice of a particular chemotherapeutic agent or other treatmentmodality such as surgery or radiation or a choice about whether towithhold or deliver therapy.

A “human consensus framework” refers to a framework which represents themost commonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

An “acceptor human framework” for the purposes herein refers to aframework comprising the amino acid sequence of a light chain variabledomain (VL) framework or a heavy chain variable domain (VH) frameworkderived from a human immunoglobulin framework or a human consensusframework, as defined below. An acceptor human framework “derived from”a human immunoglobulin framework or a human consensus framework maycomprise the same amino acid sequence thereof, or it may contain aminoacid sequence changes. In some embodiments, the number of amino acidchanges are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 orless, 4 or less, 3 or less, or 2 or less. In some embodiments, the VLacceptor human framework is identical in sequence to the VL humanimmunoglobulin framework sequence or human consensus framework sequence.

“Antigen-binding site” refers to the interface formed by one or morecomplementary determining regions. An antibody molecule has two antigencombining sites, each containing portions of a heavy chain variableregion and portions of a light chain variable region. The antigencombining sites can contain other portions of the variable regiondomains in addition to the CDRs.

An “antibody light chain” or an “antibody heavy chain” refers to apolypeptide comprising the VL or VH, respectively. The VL is encoded bythe minigenes V (variable) and J (junctional), and the VH by minigenesV, D (diversity), and J. Each of VL or VH includes the CDRs as well asthe framework regions. In this application, antibody light chains and/orantibody heavy chains may, from time to time, be collectively referredto as “antibody chains.” These terms encompass antibody chainscontaining mutations that do not disrupt the basic structure of VL orVH, as one skilled in the art will readily recognize.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide bonded. From N- to C-terminus, each heavy chain has a variableregion (V H), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(V L), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (K) andlambda (K), based on the amino acid sequence of its constant domain.

“Combinatorial library” refers to collections of compounds formed byreacting different combinations of interchangeable chemical “buildingblocks” to produce a collection of compounds based on permutations ofthe building blocks. For an antibody combinatorial library, the buildingblocks are the component V, D and J regions (or modified forms thereof)from which antibodies are formed. For purposes herein, the terms“library” or “collection” are used interchangeably.

A “combinatorial antibody library” refers to a collection of antibodies(or portions thereof, such as Fabs), where the antibodies are encoded bynucleic acid molecules produced by the combination of V, D and J genesegments, particularly human V, D and J germline segments. Thecombinatorial libraries herein typically contain at least 50 differentantibody (or antibody portions or fragment) members, typically at orabout 50, 100, 500, 103, 1×103, 2×103, 3×103, 4×103, 5×103, 6×103,7×103, 8×103, 9×103, 1×104, 2×104, 3×104, 4×104, 5×104, 6×104, 7×104,8×104, 9×104, 1×105, 2×105, 3×105, 4×105, 5×105, 6×105, 7×105, 8×105,9×105, 106, 107, 108, 109, 1010, or more different members. Theresulting libraries or collections of antibodies or portions thereof,can be screened for binding to a target protein or modulation of afunctional activity.

A “human combinatorial antibody library” refers to a collection ofantibodies or portions thereof, whereby each member contains a VL and VHchains or a sufficient portion thereof to form an antigen binding siteencoded by nucleic acid containing human germline segments produced asdescribed herein.

A “variable germline segment” refers to V, D and J groups, subgroups,genes or alleles thereof. Gene segment sequences are accessible fromknown database (e.g., National Center for Biotechnology Information(NCBI), the international ImMunoGeneTics Information System® (IMGT), theKabat database and the Tomlinson's VBase database (Lefranc (2003)Nucleic Acids Res., 31:307-310; Martin et al., Bioinformatics Tools forAntibody Engineering in Handbook of Therapeutic Antibodies, Wiley-VCH(2007), pp. 104-107). Tables 3-5 list exemplary human variable germlinesegments. Sequences of exemplary VH, DH, JH, Vκ, Jκ, Vλ and or Jλ,germline segments are set forth in SEQ ID NOS: 10-451 and 868. Forpurposes herein, a germline segment includes modified sequences thereof,that are modified in accord with the rules of sequence compilationprovided herein to permit practice of the method. For example, germlinegene segments include those that contain one amino acid deletion orinsertion at the 5′ or 3′ end compared to any of the sequences ofnucleotides set forth in SEQ ID NOS:10-451, 868.

“Compilation,” “compile,” “combine,” “combination,” “rearrange,”“rearrangement,” or other similar terms or grammatical variationsthereof refers to the process by which germline segments are ordered orassembled into nucleic acid sequences representing genes. For example,variable heavy chain germline segments are assembled such that the VHsegment is 5′ to the DH segment which is 5′ to the JH segment, therebyresulting in a nucleic acid sequence encoding a VH chain. Variable lightchain germline segments are assembled such that the VL segment is 5′ tothe JL segment, thereby resulting in a nucleic acid sequence encoding aVL chain. A constant gene segment or segments also can be assembled ontothe 3′ end of a nucleic acid encoding a VH or VL chain.

“Linked,” or “linkage” or other grammatical variations thereof withreference to germline segments refers to the joining of germlinesegments. Linkage can be direct or indirect. Germline segments can belinked directly without additional nucleotides between segments, oradditional nucleotides can be added to render the entire segmentin-frame, or nucleotides can be deleted to render the resulting segmentin-frame. It is understood that the choice of linker nucleotides is madesuch that the resulting nucleic acid molecule is in-frame and encodes afunctional and productive antibody.

“In-frame” or “linked in-frame” with reference to linkage of humangermline segments means that there are insertions and/or deletions inthe nucleotide germline segments at the joined junctions to render theresulting nucleic acid molecule in-frame with the 5′ start codon (ATG),thereby producing a “productive” or functional full-length polypeptide.The choice of nucleotides inserted or deleted from germline segments,particularly at joints joining various VD, DJ and VJ segments, is inaccord with the rules provided in the method herein for V(D)J jointgeneration. For example, germline segments are assembled such that theVH segment is 5′ to the DH segment which is 5′ to the JH segment. At thejunction joining the VH and the DH and at the junction joining the DHand JH segments, nucleotides can be inserted or deleted from theindividual VH, DH or JH segments, such that the resulting nucleic acidmolecule containing the joined VDJ segments are in-frame with the 5′start codon (ATG).

A portion of an antibody includes sufficient amino acids to form anantigen binding site.

A “reading frame” refers to a contiguous and non-overlapping set ofthree-nucleotide codons in DNA or RNA. Because three codons encode oneamino acid, there exist three possible reading frames for givennucleotide sequence, reading frames 1, 2 or 3. For example, the sequenceACTGGTCA will be ACT GGT CA for reading frame 1, A CTG GTC A for readingframe 2 and AC TGG TCA for reading frame 3. Generally for practice ofthe method described herein, nucleic acid sequences are combined so thatthe V sequence has reading frame 1.

A “stop codon” refers to a three-nucleotide sequence that signals a haltin protein synthesis during translation, or any sequence encoding thatsequence (e.g. a DNA sequence encoding an RNA stop codon sequence),including the amber stop codon (UAG or TAG)), the ochre stop codon (UAAor TAA)) and the opal stop codon (UGA or TGA)). It is not necessary thatthe stop codon signal termination of translation in every cell or inevery organism. For example, in suppressor strain host cells, such asamber suppressor strains and partial amber suppressor strains,translation proceeds through one or more stop codon (e.g. the amber stopcodon for an amber suppressor strain), at least some of the time.

A “variable heavy” (VH) chain or a “variable light” (VL) chain (alsotermed VH domain or VL domain) refers to the polypeptide chains thatmake up the variable domain of an antibody. For purposes herein, heavychain germline segments are designated as VH, DH and JH, and compilationthereof results in a nucleic acid encoding a VH chain. Light chaingermline segments are designated as VL or JL, and include kappa andlambda light chains (Vκ and Jκ; Vλ and Jλ) and compilation thereofresults in a nucleic acid encoding a VL chain. It is understood that alight chain is either a kappa or lambda light chain, but does notinclude a kappa/lambda combination by virtue of compilation of a Vκ andJλ.

A “degenerate codon” refers to three-nucleotide codon that specifies thesame amino acid as a codon in a parent nucleotide sequence. One of skillin the art is familiar with degeneracy of the genetic code and canidentify degenerate codons.

“Diversity” with respect to members in a collection refers to the numberof unique members in a collection. Hence, diversity refers to the numberof different amino acid sequences or nucleic acid sequences,respectively, among the analogous polypeptide members of thatcollection. For example, a collection of polynucleotides having adiversity of 104 contains 104 different nucleic acid sequences among theanalogous polynucleotide members. In one example, the providedcollections of polynucleotides and/or polypeptides have diversities ofat least at or about 102, 103, 104, 105, 106, 107, 108, 109, 1010 ormore.

“Sequence diversity” refers to a representation of nucleic acid sequencesimilarity and is determined using sequence alignments, diversityscores, and/or sequence clustering. Any two sequences can be aligned bylaying the sequences side-by-side and analyzing differences withinnucleotides at every position along the length of the sequences.Sequence alignment can be assessed in silico using Basic Local AlignmentSearch Tool (BLAST), an NCBI tool for comparing nucleic acid and/orprotein sequences. The use of BLAST for sequence alignment is well knownto one of skill in the art. The Blast search algorithm compares twosequences and calculates the statistical significance of each match (aBlast score). Sequences that are most similar to each other will have ahigh Blast score, whereas sequences that are most varied will have a lowBlast score.

A “polypeptide domain” refers to a part of a polypeptide (a sequence ofthree or more, generally 5 or 7 or more amino acids) that is astructurally and/or functionally distinguishable or definable. Exemplaryof a polypeptide domain is a part of the polypeptide that can form anindependently folded structure within a polypeptide made up of one ormore structural motifs (e.g. combinations of alpha helices and/or betastrands connected by loop regions) and/or that is recognized by aparticular functional activity, such as enzymatic activity or antigenbinding. A polypeptide can have one, typically more than one, distinctdomains. For example, the polypeptide can have one or more structuraldomains and one or more functional domains. A single polypeptide domaincan be distinguished based on structure and function. A domain canencompass a contiguous linear sequence of amino acids. Alternatively, adomain can encompass a plurality of non-contiguous amino acid portions,which are non-contiguous along the linear sequence of amino acids of thepolypeptide. Typically, a polypeptide contains a plurality of domains.For example, each heavy chain and each light chain of an antibodymolecule contains a plurality of immunoglobulin (Ig) domains, each about110 amino acids in length.

An “Ig domain” refers to a domain, recognized as such by those in theart, that is distinguished by a structure, called the Immunoglobulin(Ig) fold, which contains two beta-pleated sheets, each containinganti-parallel beta strands of amino acids connected by loops. The twobeta sheets in the Ig fold are sandwiched together by hydrophobicinteractions and a conserved intra-chain disulfide bond. Individualimmunoglobulin domains within an antibody chain further can bedistinguished based on function. For example, a light chain contains onevariable region domain (VL) and one constant region domain (CL), while aheavy chain contains one variable region domain (VH) and three or fourconstant region domains (CH). Each VL, CL, VH, and CH domain is anexample of an immunoglobulin domain.

A “variable domain” with reference to an antibody refers to a specificIg domain of an antibody heavy or light chain that contains a sequenceof amino acids that varies among different antibodies. Each light chainand each heavy chain has one variable region domain (VL, and, VH). Thevariable domains provide antigen specificity, and thus are responsiblefor antigen recognition. Each variable region contains CDRs that arepart of the antigen binding site domain and framework regions (FRs).

A “constant region domain” refers to a domain in an antibody heavy orlight chain that contains a sequence of amino acids that iscomparatively more conserved among antibodies than the variable regiondomain. Each light chain has a single light chain constant region (CL)domain and each heavy chain contains one or more heavy chain constantregion (CH) domains, which include, CH1, CH2, CH3 and CH4. Full-lengthIgA, IgD and IgG isotypes contain CH1, CH2 CH3 and a hinge region, whileIgE and IgM contain CH1, CH2 CH3 and CH4. CH1 and CL domains extend theFab arm of the antibody molecule, thus contributing to the interactionwith antigen and rotation of the antibody arms. Antibody constantregions can serve effector functions, such as, but not limited to,clearance of antigens, pathogens and toxins to which the antibodyspecifically binds, e.g. through interactions with various cells,biomolecules and tissues.

An “antibody or portion thereof that is sufficient to form an antigenbinding site” means that the antibody or portion thereof contains atleast 1 or 2, typically 3, 4, 5 or all 6 CDRs of the VH and VLsufficient to retain at least a portion of the binding specificity ofthe corresponding full-length antibody containing all 6 CDRs. Generally,a sufficient antigen binding site at least requires CDR3 of the heavychain (CDRH3). It typically further requires the CDR3 of the light chain(CDRL3). As described herein, one of skill in the art knows and canidentify the CDRs based on Kabat or Chothia numbering (see, e.g., Kabat,E. A. et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol.196:901-917). For example, based on Kabat numbering, CDR-L1 correspondsto residues L24-L34; CDR-L2 corresponds to residues L50-L56; CDR-L3corresponds to residues L89-L97; CDR-H1 corresponds to residues H31-H35,35a or 35b depending on the length; CDR-H2 corresponds to residuesH50-H65; and CDR-H3 corresponds to residues H95-H102.

A “peptide mimetic” refers to a peptide that mimics the activity of apolypeptide. For example, an erythropoietin (EPO) peptide mimetic is apeptide that mimics the activity of Epo, such as for binding andactivation of the EPO receptor.

An “address” refers to a unique identifier for each locus in acollection whereby an addressed member (e.g. an antibody) can beidentified. An addressed moiety is one that can be identified by virtueof its locus or location. Addressing can be effected by position on asurface, such as a well of a microplate. For example, an address for aprotein in a microwell plate that is F9 means that the protein islocated in row F, column 9 of the microwell plate. Addressing also canbe effected by other identifiers, such as a tag encoded with a bar codeor other symbology, a chemical tag, an electronic, such RF tag, acolor-coded tag or other such identifier.

An “array” refers to a collection of elements, such as antibodies,containing three or more members.

A “spatial array” refers to an array where members are separated oroccupy a distinct space in an array. Hence, spatial arrays are a type ofaddressable array. Examples of spatial arrays include microtiter plateswhere each well of a plate is an address in the array. Spacial arraysinclude any arrangement wherein a plurality of different molecules,e.g., polypeptides, are held, presented, positioned, situated, orsupported. Arrays can include microtiter plates, such as 48-well,96-well, 144-well, 192-well, 240-well, 288-well, 336-well, 384-well,432-well, 480-well, 576-well, 672-well, 768-well, 864-well, 960-well,1056-well, 1152-well, 1248-well, 1344-well, 1440-well, or 1536-wellplates, tubes, slides, chips, flasks, or any other suitable laboratoryapparatus. Furthermore, arrays can also include a plurality ofsub-arrays. A plurality of sub-arrays encompasses an array where morethan one arrangement is used to position the polypeptides. For example,multiple 96-well plates could constitute a plurality of sub-arrays and asingle array.

An “addressable library” or “spatially addressed library” refers to acollection of molecules such as nucleic acid molecules or proteinagents, such as antibodies, in which each member of the collection isidentifiable by virtue of its address.

An “addressable array” refers to one in which the members of the arrayare identifiable by their address, the position in a spatial array, suchas a well of a microtiter plate, or on a solid phase support, or byvirtue of an identifiable or detectable label, such as by color,fluorescence, electronic signal (i.e. RF, microwave or other frequencythat does not substantially alter the interaction of the molecules ofinterest), bar code or other symbology, chemical or other such label.Hence, in general the members of the array are located at identifiableloci on the surface of a solid phase or directly or indirectly linked toor otherwise associated with the identifiable label, such as affixed toa microsphere or other particulate support (herein referred to as beads)and suspended in solution or spread out on a surface.

“An addressable combinatorial antibody library” refers to a collectionof antibodies in which member antibodies are identifiable and allantibodies with the same identifier, such as position in a spatial arrayor on a solid support, or a chemical or RF tag, bind to the sameantigen, and generally are substantially the same in amino acidsequence. For purposes herein, reference to an “addressable arrayedcombinatorial antibody library” means that the antibody members areaddressed in an array.

“In silico” refers to research and experiments performed using acomputer. In silico methods include, but are not limited to, molecularmodeling studies, biomolecular docking experiments, and virtualrepresentations of molecular structures and/or processes, such asmolecular interactions. For purposes herein, the antibody members of alibrary can be designed using a computer program that selects componentV, D and J germline segments from among those input into the computerand joins them in-frame to output a list of nucleic acid molecules forsynthesis. Thus, the recombination of the components of the antibodiesin the collections or libraries provided herein, can be performed insilico by combining the nucleotide sequences of each building block inaccord with software that contains rules for doing so. The process couldbe performed manually without a computer, but the computer provides theconvenience of speed.

A “database” refers to a collection of data items. For purposes herein,reference to a database is typically with reference to antibodydatabases, which provide a collection of sequence and structureinformation for antibody genes and sequences. Exemplary antibodydatabases include, but are not limited to, IMGT®, the internationalImMunoGeneTics information system (imgt.cines.fr; see e.g., Lefranc etal. (2008) Briefings in Bioinformatics, 9:263-275), National Center forBiotechnology Information (NCBI), the Kabat database and the Tomlinson'sVBase database (Lefranc (2003) Nucleic Acids Res., 31:307-310; Martin etal., Bioinformatics Tools for Antibody Engineering in Handbook ofTherapeutic Antibodies, Wiley-VCH (2007), pp. 104-107). A database alsocan be created by a user to include any desired sequences. The databasecan be created such that the sequences are inputted in a desired format(e.g., in a particular reading frame; lacking stop codons; lackingsignal sequences). The database also can be created to include sequencesin addition to antibody sequences.

“Screening” refers to identification or selection of an antibody orportion thereof from a collection or library of antibodies and/orportions thereof, based on determination of the activity or property ofan antibody or portion thereof. Screening can be performed in any of avariety of ways, including, for example, by assays assessing directbinding (e.g. binding affinity) of the antibody to a target protein orby functional assays assessing modulation of an activity of a targetprotein.

“Activity towards a target protein” refers to binding specificity and/ormodulation of a functional activity of a target protein, or othermeasurements that reflects the activity of an antibody or portionthereof towards a target protein.

A “target protein” or “protein target” refers to candidate proteins orpeptides that are specifically recognized by an antibody or portionthereof and/or whose activity is modulated by an antibody or portionthereof. Modulating the activity can comprise increasing, decreasing,stimulating, or preventing the activity or expression of the targetprotein. A target protein includes any peptide or protein that containsan epitope for antibody recognition. Target proteins include proteinsinvolved in the etiology of a disease or disorder by virtue ofexpression or activity. Exemplary target proteins are described herein.In some instances, the target protein is a transmembrane protein target.Transmembrane protein targets include, but are not limited to, GPCRs,ion channels, transporters, and cell surface receptors. Ion channels maybe potassium ion channels, sodium ion channels, calcium ion channels,and voltage gated channels. In some instances, the antibodies disclosedherein modulate a Kv1.3 ion channel, Nav1.7 ion channel, or acid sensingion channel (ASIC). The antibodies disclosed herein may modulate cellsurface receptors such as GLP1R, GCGR, EPO receptor, FGFR, FGF21R, CSFR,GMCSFR, and GCSFR. Additional target proteins include, but are notlimited to, cytokines, kinases, interferons, hormones, and growthfactors. The target proteins can be from a mammal or non-mammal. Thetarget proteins can be from a human. Alternatively, the target proteinsare from a bovine.

“Hit” refers to an antibody or portion thereof identified, recognized orselected as having an activity in a screening assay.

“Iterative” with respect to screening means that the screening isrepeated a plurality of times, such as 2, 3, 4, 5 or more times, until a“Hit” is identified whose activity is optimized or improved compared toprior iterations.

“High-throughput” refers to a large-scale method or process that permitsmanipulation of large numbers of molecules or compounds, generally tensto hundreds to thousands of compounds. For example, methods ofpurification and screening can be rendered high-throughput.High-throughput methods can be performed manually. Generally, however,high-throughput methods involve automation, robotics or software.

Basic Local Alignment Search Tool (BLAST) is a search algorithmdeveloped by Altschul et al. (1990) to separately search protein or DNAdatabases, for example, based on sequence identity. For example, blastnis a program that compares a nucleotide query sequence against anucleotide sequence database (e.g. GenBank). BlastP is a program thatcompares an amino acid query sequence against a protein sequencedatabase.

A BLAST bit score is a value calculated from the number of gaps andsubstitutions associated with each aligned sequence. The higher thescore, the more significant the alignment.

A “human protein” refers to a protein encoded by a nucleic acidmolecule, such as DNA, present in the genome of a human, including allallelic variants and conservative variations thereof. A variant ormodification of a protein is a human protein if the modification isbased on the wildtype or prominent sequence of a human protein.

“Naturally occurring amino acids” refer to the 20 L-amino acids thatoccur in polypeptides. The residues are those 20 α-amino acids found innature which are incorporated into protein by the specific recognitionof the charged tRNA molecule with its cognate mRNA codon in humans.

“Non-naturally occurring amino acids” refer to amino acids that are notgenetically encoded. For example, a non-natural amino acid is an organiccompound that has a structure similar to a natural amino acid but hasbeen modified structurally to mimic the structure and reactivity of anatural amino acid. Non-naturally occurring amino acids thus include,for example, amino acids or analogs of amino acids other than the 20naturally-occurring amino acids and include, but are not limited to, theD-isostereomers of amino acids. Exemplary non-natural amino acids areknown to those of skill in the art.

“Nucleic acids” include DNA, RNA and analogs thereof, including peptidenucleic acids (PNA) and mixtures thereof. Nucleic acids can be single ordouble-stranded. When referring to probes or primers, which areoptionally labeled, such as with a detectable label, such as afluorescent or radiolabel, single-stranded molecules are contemplated.Such molecules are typically of a length such that their target isstatistically unique or of low copy number (typically less than 5,generally less than 3) for probing or priming a library. Generally aprobe or primer contains at least 14, 16 or 30 contiguous nucleotides ofsequence complementary to or identical to a gene of interest. Probes andprimers can be 10, 20, 30, 50, 100 or more nucleic acids long.

A “peptide” refers to a polypeptide that is from 2 to 40 amino acids inlength.

The amino acids which occur in the various sequences of amino acidsprovided herein are identified according to their known, three-letter orone-letter abbreviations (Table 1). The nucleotides which occur in thevarious nucleic acid fragments are designated with the standardsingle-letter designations used routinely in the art.

An “amino acid” is an organic compound containing an amino group and acarboxylic acid group. A polypeptide contains two or more amino acids.For purposes herein, amino acids include the twenty naturally-occurringamino acids, non-natural amino acids and amino acid analogs (i.e., aminoacids wherein the α-carbon has a side chain).

“Amino acid residue” refers to an amino acid formed upon chemicaldigestion (hydrolysis) of a polypeptide at its peptide linkages. Theamino acid residues described herein are presumed to be in the “L”isomeric form. Residues in the “D” isomeric form, which are sodesignated, can be substituted for any L-amino acid residue as long asthe desired functional property is retained by the polypeptide. NH2refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature described in J. Biol. Chem., 243: 3552-3559 (1969), andadopted 37 C.F.R. □§§1.821-1.822, abbreviations for amino acid residuesare shown below:

SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr Tyrosine G Gly Glycine F PhePhenylalanine M Met Methionine A Ala Alanine S Ser Serine I IleIsoleucine L Leu Leucine T Thr Threonine V Val Valine P Pro Proline KLys Lysine H His Histidine Q Gln Glutamine E Glu Glutamic acid Z Glx Gluand/or Gln W Trp Tryptophan R Arg Arginine D Asp Aspartic acid N AsnAsparagine B Asx Asn and/or Asp C Cys Cysteine X Xaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left to right orientation in the conventionaldirection of amino-terminus to carboxyl-terminus. In addition, thephrase “amino acid residue” is broadly defined to include the aminoacids listed in the Table of Correspondence (Table 1) and modified andunusual amino acids, such as those referred to in 37 C.F.R.§§1.821-1.822, and incorporated herein by reference. Furthermore, itshould be noted that a dash at the beginning or end of an amino acidresidue sequence indicates a peptide bond to a further sequence of oneor more amino acid residues, to an amino-terminal group such as NH2 orto a carboxyl-terminal group such as COOH. The abbreviations for anyprotective groups, amino acids and other compounds, are, unlessindicated otherwise, in accord with their common usage, recognizedabbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature(see, (1972) Biochem. 11:1726). Each naturally occurring L-amino acid isidentified by the standard three letter code (or single letter code) orthe standard three letter code (or single letter code) with the prefix“L-”; the prefix “D-” indicates that the stereoisomeric form of theamino acid is D.

An “immunoconjugate” refers to an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent, non-antibody peptide or therapeutic polypeptide. Animmunoconjugate may include non-antibody sequences. The non-antibodysequence can be conjugated to the antibody. Alternatively, thenon-antibody sequence can be within the antibody sequence.

A “non-antibody peptide” refers to a peptide encoded by a non-antibodyantibody sequence. For example, a non-antibody peptide may be a hormone,a lymphokine, an interleukin, a chemokines, a cytokine or a peptidetoxin.

As used herein, the terms “therapeutic polypeptide,” “therapeuticpeptides,” and therapeutic immunoglobulin construct” mean one or morepeptides having demonstrated or potential use in treating, preventing,or ameliorating one or more diseases, disorders, or conditions in asubject in need thereof, as well as related peptides. Therapeuticpeptides include peptides found to have use in treating, preventing, orameliorating one or more diseases, disorders, or conditions after thetime of filing of this application. Related peptides include fragmentsof therapeutic peptides, therapeutic peptide variants, and therapeuticpeptide derivatives that retain some or all of the therapeuticactivities of the therapeutic peptide. As will be known to one of skillin the art, as a general principle, modifications may be made topeptides that do not alter, or only partially abrogate, the propertiesand activities of those peptides. In some instances, modificationsresult in an increase in therapeutic activities. The terms “therapeuticpolypeptide” or “therapeutic peptides” encompass modifications to thetherapeutic peptides defined and/or disclosed herein. In certainembodiments, the therapeutic polypeptide is selected from a hormone, alymphokine, an interleukin, a chemokines, a cytokine, a peptide toxin,and combinations thereof. Therapeutic polypeptides can be peptidesencoded by non-antibody sequences.

A derivative or a variant of a polypeptide is said to share “homology”or be “homologous” with the peptide if the amino acid sequences of thederivative or variant has at least 50% identity with the originalpeptide. In certain embodiments, the derivative or variant is at least75% the same as that of either the peptide or a fragment of the peptidehaving the same number of amino acid residues as the derivative. Incertain embodiments, the derivative or variant is at least 85% the sameas that of either the peptide or a fragment of the peptide having thesame number of amino acid residues as the derivative. In certainembodiments, the amino acid sequence of the derivative is at least 90%the same as the peptide or a fragment of the peptide having the samenumber of amino acid residues as the derivative. In some embodiments,the amino acid sequence of the derivative is at least 95% the same asthe peptide or a fragment of the peptide having the same number of aminoacid residues as the derivative. In certain embodiments, the derivativeor variant is at least 99% the same as that of either the peptide or afragment of the peptide having the same number of amino acid residues asthe derivative.

Groupings of alternative elements or embodiments disclosed herein arenot to be construed as limitations. Each group member can be referred toand claimed individually or in any combination with other members of thegroup or other elements found herein. It is anticipated that one or moremembers of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Certain embodiments are described herein, including the best mode knownto the inventors for carrying out the exemplary embodiments. Of course,variations on these described embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventor expects skilled artisans to employ such variations asappropriate, and the inventors intend for the embodiments to bepracticed otherwise than specifically described herein. Accordingly,this disclosure includes all modifications and equivalents of thesubject matter recited in the claims appended hereto as permitted byapplicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

Furthermore, numerous references have been made to patents and printedpublications. Each of the above-cited references is individuallyincorporated herein by reference in their entirety.

Specific embodiments disclosed herein can be further limited in theclaims using consisting of or and consisting essentially of language.When used in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Exemplary embodiments so claimed are inherently orexpressly described and enabled herein.

In closing, it is to be understood that the exemplary embodimentsdisclosed herein are illustrative of the principles of the presentdisclosure. Other modifications that can be employed are within thescope of the disclosure. Thus, by way of example, but not of limitation,alternative configurations of the present exemplary embodiments can beutilized in accordance with the teachings herein. Accordingly, thepresent exemplary embodiments are not limited to that precisely as shownand described.

General Techniques

The present disclosure relies on routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this present disclosure include Sambrook and Russell, MolecularCloning: A Laboratory Manual 3d ed. (2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Ausubel et al., CurrentProtocols in Molecular Biology (1994).

For nucleic acids, sizes are given in either kilobases (Kb) or basepairs (bp). These are estimates derived from agarose or polyacrylamidegel electrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilo-Daltons (kD) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage and Caruthers, Tetrahedron Letters,22:1859-1862 (1981), using an automated synthesizer, as described in VanDevanter et al., Nucleic Acids Res., 12:6159-6168 (1984). Purificationof oligonucleotides is by either native polyacrylamide gelelectrophoresis or by anion-exchange chromatography as described inPearson & Reanier, J. Chrom., 255:137-149 (1983). The sequence of thecloned genes and synthetic oligonucleotides can be verified aftercloning using, e.g., the chain termination method for sequencingdouble-stranded templates of Wallace et al., Gene, 16:21-26 (1981).

The nucleic acids encoding recombinant polypeptides of the presentdisclosure may be cloned into an intermediate vector beforetransformation into prokaryotic or eukaryotic cells for replicationand/or expression. The intermediate vector may be a prokaryote vectorsuch as a plasmid or shuttle vector.

Antibodies with Ultralong CDR3 Sequences

To date, cattle are the only species where ultralong CDR3 sequences havebeen identified. However, other species, for example other ruminants,may also possess antibodies with ultralong CDR3 sequences.

Exemplary antibody variable region sequences comprising an ultralongCDR3 sequence identified in cattle include those designated as: BLV1H12(see, SEQ ID NO: 22), BLV5B8 (see, SEQ ID NO: 23), BLV5D3 (see, SEQ IDNO: 24) and BLV8C11 (see, SEQ ID NO: 25) (see, e.g., Saini, et al.(1999) Eur. J. Immunol. 29: 2420-2426; and Saini and Kaushik (2002)Scand. J. Immunol. 55: 140-148); BF4E9 (see, SEQ ID NO: 26) and BF1H1(see, SEQ ID NO: 27) (see, e.g., Saini and Kaushik (2002) Scand. J.Immunol. 55: 140-148); and F18 (see, SEQ ID NO: 28) (see, e.g., Berens,et al. (1997) Int. Immunol. 9: 189-199).

In an embodiment, bovine antibodies are identified and/or produced.Multiple techniques exist to identify and/or produce antibodies.

Antibodies of the present disclosure may be isolated by screeningincluding, high-throughput screening, of combinatorial libraries forantibodies with the desired activity or activities. For example, avariety of methods are known in the art for generating phage displaylibraries and screening such libraries for antibodies possessing thedesired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).Such screening may be iterative until a hit is obtained.

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Phage displaylibraries of bovine antibodies may be a source of bovine antibody genesequences, including ultralong CDR3 sequences.

Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which CDRs (or portions thereof) are derivedfrom a non-human antibody, and FRs (or portions thereof) are derivedfrom human antibody sequences. A humanized antibody optionally will alsocomprise at least a portion of a human constant region. In someembodiments, some FR residues in a humanized antibody are substitutedwith corresponding residues from a non-human antibody (e.g., theantibody from which the CDR residues are derived), e.g., to restore orimprove antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005); Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling); and Studnicka et al., U.S. Pat. No. 5,766,886.

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al.,Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

Antibodies with ultralong CDR3 sequences may also include non-antibodysequences, such as cytokines, therapeutic polypeptides or growthfactors, into the CDR3 region. The resultant antibody can be effectivein treating or preventing a disease or condition. For example, anantibody comprising an ultralong CDR3 inhibits tumor metastasis. In someembodiments, the cytokine, therapeutic polypeptide or growth factor maybe shown to have an antiproliferative effect on at least one cellpopulation. Alternatively, or additionally, the resultant antibodymodulates the expression or activity of a target (e.g., protein target,transmembrane protein target). For example, an antibody comprising anultralong CDR3 inhibits or blocks an ion channel. The non-antibodysequence may be a hormone, a lymphokine, an interleukin, a chemokines, acytokine, a peptide toxin, and combinations thereof. Such cytokines,therapeutic polypeptides, toxins, lymphokines, growth factors, or otherhematopoietic factors include Granulocyte colony-stimulating factor(G-CSF), macrophage colony-stimulating factor (M-CSF),Granulocyte-macrophage colony-stimulating factor (GM-CSF), Meg-CSF, TNF,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN (e.g.,α-interferon, β-interferon, a λ-interferon), TNF-alpha, TNF1, TNF2,thrombopoietin, stem cell factor, and erythropoietin (EPO). Additionalgrowth factors for use in the antibodies and/or pharmaceuticalcompositions of the present disclosure include: angiogenin, bonemorphogenic protein-1, bone morphogenic protein-2, bone morphogenicprotein-3, bone morphogenic protein-4, bone morphogenic protein-5, bonemorphogenic protein-6, bone morphogenic protein-7, bone morphogenicprotein-8, bone morphogenic protein-9, bone morphogenic protein-10, bonemorphogenic protein-11, bone morphogenic protein-12, bone morphogenicprotein-13, bone morphogenic protein-14, bone morphogenic protein-15,bone morphogenic protein receptor IA, bone morphogenic protein receptorIB, brain derived neurotrophic factor, ciliary neutrophic factor,ciliary neutrophic factor receptor, cytokine-induced neutrophilchemotactic factor 1, cytokine-induced neutrophil, chemotactic factor 2,cytokine-induced neutrophil chemotactic factor 2, endothelial cellgrowth factor, endothelin 1, epidermal growth factor, epithelial-derivedneutrophil attractant, fibroblast growth factor 4, fibroblast growthfactor 5, fibroblast growth factor 6, fibroblast growth factor 7,fibroblast growth factor 8, fibroblast growth factor 8b, fibroblastgrowth factor 8c, fibroblast growth factor 9, fibroblast growth factor10, fibroblast growth factor 21 (FGF21)fibroblast growth factor acidic,fibroblast growth factor basic, glial cell line-derived neutrophicfactor receptor-1, glial cell line-derived neutrophic factor receptor-2,growth related protein, growth related protein-1, growth relatedprotein-2, growth related protein-3, heparin binding epidermal growthfactor, hepatocyte growth factor, hepatocyte growth factor receptor,insulin-like growth factor I, insulin-like growth factor receptor,insulin-like growth factor II, insulin-like growth factor bindingprotein, keratinocyte growth factor, leukemia inhibitory factor,leukemia inhibitory factor receptor-1, nerve growth factor nerve growthfactor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor,placenta growth factor 2, platelet-derived endothelial cell growthfactor, platelet derived growth factor, platelet derived growth factor Achain, platelet derived growth factor AA, platelet derived growth factorAB, platelet derived growth factor B chain, platelet derived growthfactor BB, platelet derived growth factor receptor-1, platelet derivedgrowth factor receptor-2, pre-B cell growth stimulating factor, stemcell factor, stem cell factor receptor, transforming growth factor-1,transforming growth factor-2, transforming growth factor-1, transforminggrowth factor-1.2, transforming growth factor-2, transforming growthfactor-3, transforming growth factor-S, latent transforming growthfactor-1, transforming growth factor-1 binding protein I, transforminggrowth factor-1 binding protein II, transforming growth factor-1 bindingprotein III, tumor necrosis factor receptor type I, tumor necrosisfactor receptor type II, urokinase-type plasminogen activator receptor,vascular endothelial growth factor, and chimeric proteins andbiologically or immunologically active fragments thereof. In someembodiments, the therapeutic polypeptide is a mammalian G-CSF, a growthhormone, a leptin, a α-interferon, a β-interferon, a λ-interferon, aGM-CSF, a IL-11, a IL-10, a mokal (e.g., Moka, mokatoxin-1), or a VM-24.In some embodiments, the therapeutic polypeptide is a glucagon-likepeptide 1 (GLP-1), exendin-4 (Ex-4), erythropoietin (EPO), fibroblastgrowth factor (FGF21), IL8, ziconotide, somatostatin, chlorotoxin,SDF1(alpha), IL21, or derivative or variant thereof. The G-CSF may be abovine G-CSF. The G-CSF, GM-CSF, EPO, FGF21, β-interferon and GLP-1 maybe from a human.

The non-antibody sequence may comprise an amino acid sequence based onor derived from any of SEQ ID NOS: 317-332. The non-antibody sequencemay comprise an amino acid sequence that is 50%, 60%, 70% 80%, 90%, 95%,97%, 99% identical to any of SEQ ID NOS: 317-332. The non-antibodysequence may comprise an amino acid sequence that comprises 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acidresidues of SEQ ID NOS: 317-332. The amino acid residues may beconsecutive. Alternatively, the amino acid residues are non-consecutive.The non-antibody sequence may comprise at least a portion of any of SEQID NOS: 317-332.

The antibodies disclosed herein may comprise one or more sequences basedon or derived from a mammalian, avian, reptilian, amphibian, fish,insect, bug, or arachnid sequence. Mammals include, but are not limitedto, cows, bison, buffalo, humans, mice, dogs, cats, sheep, goats, orrabbits. Avians include, but are not limited to, chicken, geese, doves,eagles, sparrows, and pidgeons. Reptiles include, but are not limitedto, lizards, gators, snakes, and turtles. Amphibians include, but arenot limited to, frogs, salamanders, toads, and newts. Fish include, butare not limited to, tuna, salmon, whales, and sharks. Insects, bugs, andarachnids include, but are not limited to, flies, mosquitos, spiders,and scorpions. The non-antibody sequence may be based on or derived froma bovine or human sequence. Alternatively, the non-antibody sequence isbased on or derived from a lizard, snail, snake or scorpion sequence.The lizard may be a gila monster. The snail may be a cone snail.

In some embodiments, the non-antibody sequence is linked to an end of anultralong CDR3 sequence. For example, the non-antibody sequence can belinked to the 5′ end or 3′ end of the ultralong CDR3 nucleotidesequence. In another example, the non-antibody sequence can be linked tothe N-terminus or C-terminus of the ultralong CDR3 peptide sequence.

In another embodiment, the non-antibody sequence is inserted within anultralong CDR3 sequence. For example, the non-antibody sequence isinserted between the stalk domain of an ultralong CDR3 sequence. Thenon-antibody sequence can be inserted within the stalk domain of anultralong CDR3 sequence. In another example, the non-antibody sequenceis inserted between the stalk domain and the knob domain of an ultralongCDR3 sequence. Alternatively, the non-antibody sequence is insertedwithin the knob domain of an ultralong CDR3 sequence.

In some embodiments, the non-antibody sequence replaces at least aportion of an ultralong CDR3 sequence. The non-antibody sequence canreplace about 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40or more, 45 or more, 50 or more, 55 or more amino acids of the ultralongCDR3 peptide sequence. The non-antibody sequence can replace about 10%or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% ormore, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more,85% or more, 90% or more, 95% or more of the ultralong CDR3 peptidesequence. The non-antibody sequence can replace at least a portion of aknob domain of an ultralong CDR3. The non-antibody sequence can replaceabout 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 ormore, 35 or more, 40 or more, 45 or more amino acids of the knob domainof an ultralong CDR3 peptide sequence. The non-antibody sequence canreplace about 10% or more, 20% or more, 30% or more, 40% or more, 50% ormore, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more,80% or more, 85% or more, 90% or more, 95% or more of the knob domain ofthe ultralong CDR3 peptide sequence. The non-antibody sequence canreplace at least a portion of a stalk domain of an ultralong CDR3. Thenon-antibody sequence can replace about 1 or more, 2 or more, 3 or more,4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 ormore, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 ormore amino acids of the stalk domain of an ultralong CDR3 peptidesequence. The non-antibody sequence can replace about 10% or more, 20%or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% ormore, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more,90% or more, 95% or more of the stalk domain of the ultralong CDR3peptide sequence. The amino acids may be consecutive amino acids.Alternatively, the amino acids are non-consecutive amino acids. Theultralong CDR3 may comprise one or more conserved motifs. The conservedmotifs may be stalk domain conserved motifs as disclosed herein.Alternatively, the conserved motifs may be knob domain conserved motifsas disclosed herein.

In some embodiments, the non-antibody sequence replaces at least aportion of an ultralong CDR3 sequence. The non-antibody sequence canreplace about 5 or more, 10 or more, 15 or more, 20 or more, 25 or more,30 or more, 40 or more, 50 or more, 55 or more, 60 or more, 70 or more,80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 ormore, 140 or more, 150 or more, 160 or more, 170 or more nucleotides ofthe ultralong CDR3 nucleotide sequence. The non-antibody sequence canreplace about 10% or more, 20% or more, 30% or more, 40% or more, 50% ormore, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more,80% or more, 85% or more, 90% or more, 95% or more of the ultralong CDR3nucleotide sequence. The non-antibody sequence can replace at least aportion of a knob domain of an ultralong CDR3. The non-antibody sequencecan replace about 5 or more, 10 or more, 15 or more, 20 or more, 25 ormore, 30 or more, 40 or more, 50 or more, 55 or more, 60 or more, 70 ormore, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130or more, 140 or more nucleotides of the knob domain of an ultralong CDR3nucleotide sequence. The non-antibody sequence can replace about 10% ormore, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more,60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, 95% or more of the knob domain of the ultralong CDR3nucleotide sequence. The non-antibody sequence can replace at least aportion of a stalk domain of an ultralong CDR3. The non-antibodysequence can replace about 5 or more, 10 or more, 15 or more, 20 ormore, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 ormore, 55 or more, 60 or more, 65 or more, 70 or more nucleotides of thestalk domain of an ultralong CDR3 nucleotide sequence. The non-antibodysequence can replace about 10% or more, 20% or more, 30% or more, 40% ormore, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more,75% or more, 80% or more, 85% or more, 90% or more, 95% or more of thestalk domain of the ultralong CDR3 nucleotide sequence. The nucleotidesmay be consecutive nucleotides. Alternatively, the nucleotides arenon-consecutive nucleotides. The ultralong CDR3 may comprise one or moreconserved motifs. The conserved motifs may be stalk domain conservedmotifs as disclosed herein. Alternatively, the conserved motifs may beknob domain conserved motifs as disclosed herein.

An antibody comprising an ultralong CDR3 sequence and a non-antibodysequence may further comprise one or more cleavage sites between theultralong CDR3 sequence and the non-antibody sequence. The one or morecleavage sites may be in front of the N-terminus of the non-antibodypeptide sequence. For example, a cleavage site is inserted at theN-terminus of the non-antibody peptide sequence and at the C-terminus ofthe ultralong CDR3 peptide sequence. Alternatively, the one or morecleave sites are behind the C-terminus of the non-antibody peptidesequence. For example the cleavage site is inserted at the C-terminus ofthe non-antibody peptide sequence and at the N-terminus of the ultralongCDR3 peptide sequence. The one or more cleavage sites may flank both theN-terminus and the C-terminus of the non-antibody peptide sequence. Theone or more cleavage sites may be upstream of the non-antibodynucleotide sequence. For example, the one or more cleavage sites may beat the 5′ end of the non-antibody nucleotide sequence and at the 3′ endof the ultralong CDR3 nucleotide sequence. The one or more cleavagesites may be downstream of the non-antibody nucleotide sequence. Forexample, the one or more cleavage sites may be at the 3′ end of thenon-antibody nucleotide sequence and at the 5′ end of the ultralong CDR3nucleotide sequence. The one or more cleavage sites may flank both the5′ end and the 3′ end of the non-antibody nucleotide sequence. The oneor more cleavage sites may directly flank the non-antibody sequence. Forexample, there are zero nucleotides or amino acids between the cleavagesite sequence and the non-antibody sequence. Alternatively, the one ormore cleavage sites may indirectly flank the non-antibody sequence. Forexample, there are one or more nucleotides between the cleavage sitenucleotide sequence and the non-antibody nucleotide sequence. There maybe 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8or more, 9 or more, 10 or more, 12 or more, 15 or more, 20 or morenucleotides between the cleavage site nucleotide sequence and thenon-antibody nucleotide sequence. In another example, there are one ormore amino acids between the cleavage site peptide sequence and thenon-antibody peptide sequence. There may be 2 or more, 3 or more, 4 ormore, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more,12 or more, 15 or more, 20 or more amino acids between the cleavage sitepeptide sequence and the non-antibody peptide sequence. The cleavagesite may be adjacent to the sequence based on or derived from theultralong CDR3 sequence, linker sequence, non-antibody sequence,non-bovine sequence, or a combination thereof. The cleavage site may bebetween the sequence based on or derived from the ultralong CDR3sequence and the linker sequence. The cleavage site may be between thesequence based on or derived from the ultralong CDR3 sequence and thenon-antibody sequence. The cleavage site may be between the linkersequence and the non-antibody sequence. The cleavage site may be for aprotease. The protease may be a serine protease, threonine protease,cysteine protease, aspartate protease, or metalloprotease. The proteasemay include, but is not limited to, Factor Xa protease,chymotrypsin-like protease, trypsin-like protease, elastase-likeprotease, subtilisin-like protease, actinidain, bromelain, calpains,caspases, cathepsins, Mir1-CP, papain, HIV-1 protease, chymosin, renin,cathepsin D, pepsin, plasmepsin, nepenthesin, metalloexopeptidases, andmetalloendopeptidases. The cleavage site may be a cleavage site forFactor Xa or thrombin. For example, the cleavage site may comprise theamino acid sequence of IEGR. Alternatively, the cleavage site is for anuclease. The antibody comprising the ultralong CDR3 sequence andnon-antibody sequence may be cleaved by one or more proteases. Cleavageof the antibody by the one or more protease can result in release of oneor more ends of the non-antibody peptide from the ultralong CDR3 regionof the antibody. For example, cleavage of the antibody results inrelease of the N-terminus of the non-antibody peptide from the ultralongCDR3 region. Alternatively, cleavage of the antibody results in releaseof the C-terminus of the non-antibody peptide from the ultralong CDR3region.

The non-antibody sequence may be linked to the ultralong CDR3 sequencevia one or more linkers. The non-antibody sequence may be inserted withan ultralong CDR3 sequence. In some instances, two or more linkers areused to link the non-antibody sequence to the ultralong CDR3 sequence.The two or more linkers may comprise the same sequence. Alternatively,the two or more linkers comprise different sequences. The one or morelinker sequences may be the same length. The one or more linkersequences may be different lengths. The one or more linker sequences maybe 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9or more or 10 or more amino acids in length. The one or more linkersequences may comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more glycineresidues. The one or more linker sequences may comprise 2 or more, 3 ormore, 4 or more, or 5 or more consecutive glycine residues. The one ormore linker sequences may comprise 1 or more serine residues. The one ormore linker sequences may comprise 1 or more, 2 or more, 3 or more, 4 ormore, or 5 or more polar amino acid residues. The polar amino acidresidues may be selected from serine, threonine, asparagine, orglutamine. The polar amino acid residues may comprise uncharged sidechains. The linkers may be attached to the N-terminal, C-terminal, orboth N- and C-termini of the non-antibody peptide sequence. The linkersmay be attached to the 5′-end, 3′-end, or both the 5′- and 3′ ends ofthe non-antibody nucleotide sequence. In some embodiments, the linkermay comprise amino acid residues. Exemplary amino acid linker componentsinclude a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.Exemplary dipeptides include: valine-citrulline (vc or val-cit),alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include:glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine(gly-gly-gly). Alternatively, the linker comprises an amino acidsequence of (GGGGS). (SEQ ID NO: 339) wherein n=1 to 5. The linker maycomprise an amino acid sequence of GGGSGGGGS (SEQ ID NO: 337) orGGGGSGGGS (SEQ ID NO: 338). Amino acid residues which comprise an aminoacid linker component include those occurring naturally, as well asminor amino acids and non-naturally occurring amino acids includinganalogs, such as citrulline. Amino acid linker components can bedesigned and optimized in their selectivity for enzymatic cleavage by aparticular enzymes, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

The ultralong CDR3 may be based on or derived from a single ultralongCDR3 sequence. Alternatively, the ultralong CDR3 is based on or derivedfrom two or more ultralong CDR3 sequences. The two or more ultralongCDR3 sequences may be from the same animal. Alternatively, the two ormore ultralong CDR3 sequences are from two or more different animals.

The ultralong CDR3 may comprise at least a portion of a stalk domain ofan ultralong CDR3. The antibodies disclosed herein may comprise 1 ormore, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more 7 or more, 8or more, 9 or more, or 10 or more amino acids derived from or based onthe stalk domain of the ultralong CDR3. The antibodies disclosed herienmay comprise 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 orfewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer,10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer,4 or fewer, 3 or fewer, or 2 or fewer amino acids derived from or basedthe stalk domain of the ultralong CDR3. The amino acids may beconsecutive amino acids. Alternatively, the amino acids arenon-consecutive amino acids. The antibodies disclosed herein maycomprise a sequence that is 50% or more, 60% or more, 70% or more, 80%or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% ormore, or 100% homologous the sequence of the stalk domain of theultralong CDR3. The ultralong CDR3 may comprise one or more conservedmotifs derived from or based on a stalk domain of the ultralong CDR3.The antibodies disclosed herein may comprise 1 or more, 2 or more, 3 ormore, 4 or more, or 5 or more conserved motifs derived from or based onthe stalk domain of the ultralong CDR3. The one or more conserved motifsderived from or based on the stalk domain of the ultralong CDR3 maycomprise a sequence selected from any one of SEQ ID NOS: 157-307 and SEQID NOS: 333-336. The antibodies disclosed herein may comprise a sequencethat is 50% or more, 60% or more, 70% or more, 80% or more, 85% or more,90% or more, 95% or more, 97% or more, 99% or more, or 100% homologousto a sequence selected from any one of SEQ ID NOS: 157-224 and 235-295.The antibodies disclosed herein may comprise a sequence that is 50% ormore, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more,95% or more, 97% or more, 99% or more, or 100% homologous to a sequenceselected from any one of SEQ ID NOS: 225-227.

The one or more conserved motifs derived from or based on the stalkdomain of the ultralong CDR3 may comprise a CT(T/S)VHQ motif.Alternatively, the one or more conserved motifs derived from or based onthe stalk domain of the ultralong CDR3 comprise a CT(T/S)VHQX_(n) motif.In some instances, n is between 1 to 8, between 1 to 7, between 1 to 6,between 1 to 5, between 1 to 4, or between 1 to 3. The one or moreconserved motifs derived from or based on the stalk domain of theultralong CDR3 may comprise a CX¹ X² X³ X⁴Q motif. X¹ may be a T, S, A,or G residue. X² may be a T, S, A, P, or I residue. X³ may be a V or Kresidue. X⁴ may be an H, K, or Y residue. The one or more conservedmotifs derived from or based on the stalk domain of the ultralong CDR3may comprise an X¹ X²VHQ motif. X¹ may be a T, S, A, or G residue. X²may be a T, S, A, P or I residue. The one or more conserved motifsderived from or based on the stalk domain of the ultralong CDR3 maycomprise a CX¹ X²VHQ motif. X¹ may be a T, S, A, or G residue. X² may bea T, S, A, P or I residue. The one or more conserved motifs derived fromor based on the stalk domain of the ultralong CDR3 may comprise an X¹X²VX³Q motif. X¹ may be a T, S, A, or G residue. X² may be a T, S, A, Por I residue. X³ may be an H, Y or K residue. The one or more conservedmotifs derived from or based on the stalk domain of the ultralong CDR3may comprise a CX¹ X²VX³Q motif. X¹ may be a T, S, A, or G residue. X²may be a T, S, A, P or I residue. X³ may be an H, Y or K residue. Theone or more conserved motifs derived from or based on the stalk domainof the ultralong CDR3 may comprise an X¹ X²KKQ motif. X¹ may be a T, S,A, or G residue. X² may be a T, S, A, P or I residue. The one or moreconserved motifs derived from or based on the stalk domain of theultralong CDR3 may comprise a CX¹ X²KKQ motif. X¹ may be a T, S, A, or Gresidue. X² may be a T, S, A, P or I residue.

The one or more conserved motifs derived from or based on the stalkdomain of the ultralong CDR3 may comprise an YX¹YX² motif. X¹ may be aT, S, N, or I residue. X² may be an E or D residue. The one or moreconserved motifs derived from or based on the stalk domain of theultralong CDR3 may comprise an YX¹YX²Y motif. X¹ may be an L, S, T, or Rresidue. X² may be a T, S, N or I residue. The one or more conservedmotifs derived from or based on the stalk domain of the ultralong CDR3may comprise an YX¹YX²YX³ motif. X¹ may be an L, S, T, or R residue. X²may be a T, S, N or I residue. X³ may be an E or D residue. The one ormore conserved motifs derived from or based on the stalk domain of theultralong CDR3 may comprise an YX¹YX²YX³X⁴ motif X¹ may be an L, S, T,or R residue. X² may be a T, S, N or I residue. X³ may be an E or Dresidue. X⁴ may be an H, W, N, F, I or Y residue. The one or moreconserved motifs derived from or based on the stalk domain of theultralong CDR3 may comprise an Y(E/D)X motif X may be an H, W, N, F, Ior Y residue. The one or more conserved motifs derived from or based onthe stalk domain of the ultralong CDR3 may comprise an XY(E/D) motif Xmay be a T, S, N or I residue. The one or more conserved motifs derivedfrom or based on the stalk domain of the ultralong CDR3 may comprise anY(E/D)X¹X_(n)W motif X¹ may be an H, W, N, F, I or Y residue. In someinstances, n is between 1 to 4, between 1 to 3, or between 1 to 2. Theone or more conserved motifs derived from or based on the stalk domainof the ultralong CDR3 may comprise an Y(E/D)X¹X² X³X⁴X⁵W motif X¹ may bean H, W, N, F, I or Y residue. X² may be an Y, H, G, or N residue. X3may be a V, I, or A residue. X⁴ may be a D, N, T, or E residue. X⁵ maybe an A, V, S, or T residue.

The antibodies disclosed herein may comprise a first conserved motifderived from or based on the stalk domain of the ultralong CDR3 selectedfrom any of SEQ ID NOS: 157-234 and a second conserved motif derivedfrom or based on the stalk domain of the ultralong CDR3 selected fromany of SEQ ID NOS: 235-307 and SEQ ID NOS: 333-336. The antibodiesdisclosed herein may comprise a first conserved motif derived from orbased on the stalk domain of the ultralong CDR3 selected from a groupcomprising CT(T/S)VHQX_(n), CX¹X² X³X⁴Q, X¹X²VHQ, CX¹X² VHQ, X¹X²VX³Q,CX¹X²VX³Q, X¹X²KKQ, and CX¹X²KKQ and a second conserved motif derivedfrom or based on the stalk domain of the ultralong CDR3 selected fromthe group comprising YX¹YX², YX¹YX²Y, YX¹YX²YX³, YX¹YX²YX³X⁴, Y(E/D)X,XY(E/D), Y(E/D)X¹X_(n)W, and Y(E/D)X¹X² X³X⁴X⁵W.

The ultralong CDR3 may comprise at least a portion of a knob domain ofan ultralong CDR3. The antibodies disclosed herein may comprise 1 ormore, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more 7 or more, 8or more, 9 or more, or 10 or more amino acids derived from or based onthe knob domain of the ultralong CDR3. The antibodies disclosed herienmay comprise 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 orfewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer,10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer,4 or fewer, 3 or fewer, or 2 or fewer amino acids derived from or basedthe knob domain of the ultralong CDR3. The amino acids may beconsecutive amino acids. Alternatively, the amino acids arenon-consecutive amino acids. The antibodies disclosed herein maycomprise a sequence that is 50% or more, 60% or more, 70% or more, 80%or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% ormore, or 100% homologous the sequence of the knob domain of theultralong CDR3. The ultralong CDR3 may comprise one or more conservedmotifs derived from or based on a knob domain of the ultralong CDR3. Theantibodies disclosed herein may comprise 1 or more, 2 or more, 3 ormore, 4 or more, or 5 or more conserved motifs derived from or based onthe knob domain of the ultralong CDR3. The one or more conserved motifsderived from or based on the knob domain may comprise a cysteine motifas disclosed herein. Alternatively, or additionally, one or moreconserved motifs derived from or based on the knob domain comprises aC(P/S)DG motif.

The antibodies disclosed herein may comprise a sequence based on orderived from a mammal. The mammal may be a bovine. Alternatively, themammal is a non-bovine mammal, such as a human. The antibody sequencesmay be 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more,9 or more, 10 or more, 11 or more, 12 or more, 15 or more, 20 or more,25 or more, 30 or more, 35 or more, 40 or more 45 or more, 50 or more,55 or more, 60 or more, 65 or more 70 or more, 80 or more, 90 or more,100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 ormore, 160 or more, 170 or more, 180 or more 190 or more, 200 or more,220 or more, 230 or more, 240 or more 250 or more 260 or more, 270 ormore, 280 or more, 290 or more or 300 or more amino acids in length. Theamino acids may be consecutive amino acids. Alternatively, the aminoacids are non-consecutive amino acids.

The antibody sequences may comprise a bovine antibody sequencecomprising 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 15 or more, 20 ormore, 25 or more, 30 or more, 35 or more, 40 or more 45 or more, 50 ormore, 55 or more, 60 or more, 65 or more 70 or more, 80 or more, 90 ormore, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more,150 or more, 160 or more, 170 or more, 180 or more 190 or more, 200 ormore, 220 or more, 230 or more, 240 or more 250 or more 260 or more, 270or more, 280 or more, 290 or more or 300 or more amino acids in length.The bovine antibody may be a BLVH12, BLV5B8, BLVCV1, BLV5D3, BLV8C11,BF1H1, or F18 antibody. The antibody sequences may comprise a humanantibody sequence comprising 3 or more, 4 or more, 5 or more, 6 or more,7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 15or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more 45or more, 50 or more, 55 or more, 60 or more, 65 or more 70 or more, 80or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more,140 or more, 150 or more, 160 or more, 170 or more, 180 or more 190 ormore, 200 or more, 220 or more, 230 or more, 240 or more 250 or more 260or more, 270 or more, 280 or more, 290 or more or 300 or more aminoacids in length. The amino acids may be consecutive amino acids.Alternatively, the amino acids are non-consecutive amino acids.

The antibody sequence based on or derived from at least a portion of theultralong CDR3 can be 20 or fewer, 19 or fewer, 18 or fewer, 17 orfewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer,11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 orfewer, or 5 or fewer amino acids in length. The antibody sequence basedon or derived from at least a portion of the ultralong CDR3 may be 3 ormore, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more,10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 ormore amino acids in length. The amino acids may be consecutive aminoacids. Alternatively, the amino acids are non-consecutive amino acids.

The antibody sequence based on or derived from at least a portion of theultralong CDR3 can contain 1 or more, 2 or more, 3 or more, 4 or more, 5or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 ormore, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 50 ormore, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or morenucleic acid modifications or alterations in the nucleotide sequence ofthe ultralong CDR3 from which it is based on or derived from. Themodifications and/or alterations may comprise substitutions, deletions,and/or insertions. Substitutions may comprise replacing one nucleic acidwith another nucleic acid. The nucleic acid may be a natural nucleicacid or a non-natural nucleic acid.

The antibody sequence based on or derived from at least a portion of theultralong CDR3 can contain 1 or more, 2 or more, 3 or more, 4 or more, 5or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 ormore, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 50 ormore, or 60 or more amino acid modifications or alterations in thepeptide sequence of the ultralong CDR3 from which it is based on orderived from. The modifications and/or alterations may comprisesubstitutions, deletions, and/or insertions. Substitutions may comprisereplacing one amino acid with another amino acid. The amino acids to besubstituted may contain one or more similar features to the amino acidby which it is replaced. The features may include, but are not limitedto, size, polarity, hydrophobicity, acidity, side chain, and bondformations. The amino acid may be a natural amino acid or a non-naturalamino acid.

In certain embodiments, the half-life of an antibody described herein isgreater than the half-life of the un-conjugated therapeutic peptide orun-conjugated non-antibody peptide that is incorporated in the antibody.In some embodiments, the half-life of an antibody provided herein isgreater than 4 hours when administered to a subject. In certainembodiments, the half-life of an antibody provided herein is greaterthan 4 hours, greater than 6 hours, greater than 12 hours, greater than24 hours, greater than 36 hours, greater than 2 days, greater than 3days, greater than 4 days, greater than 5 days, greater than 6 days,greater than 7 days, greater than 8 days, greater than 9 days, greaterthan 10 days, greater than 11 days, greater than 12 days, greater than13 days, or greater than 14 days when administered to a subject. In someinstances, the subject is a mammal. In some embodiments, the subject isa mouse or a bovine. In other instances, the subject is a human. Incertain embodiments, a pharmaceutical composition comprising theantibody is administered to the subject once a day, every two days,every three days, every 4 days, every 7 days, every 10 days, every 14days, every 21 days, every 28 days, every 2 months, or every threemonths.

The antibodies may be modified or altered to reduce immunogenicity. Forexample, the sequence of a partially bovine or non-bovine antibody maybe modified or altered to reduce immunogenicity to humans. A non-humanantibody may be humanized to reduce immunogenicity to humans, whileretaining the specificity and affinity of the parental non-humanantibody. Generally, a humanized antibody comprises one or more variabledomains in which HVRs, e.g., CDRs, (or portions thereof) are derivedfrom a non-human antibody, and FRs (or portions thereof) are derivedfrom human antibody sequences. A humanized antibody optionally will alsocomprise at least a portion of a human constant region. In someembodiments, some FR residues in a humanized antibody are substitutedwith corresponding residues from a non-human antibody (e.g., theantibody from which the HVR residues are derived), e.g., to restore orimprove antibody specificity or affinity.

The antibodies comprising an ultralong CDR3 as disclosed herein arepreferably monoclonal. Also encompassed within the scope of thedisclosure are Fab, Fab′, Fab′-SH and F(ab′)² fragments of theantibodies comprising an ultralong CDR3 as provided herein. Theseantibody fragments can be created by traditional means, such asenzymatic digestion, or may be generated by recombinant techniques. Suchantibody fragments may be chimeric or humanized. These fragments areuseful for the diagnostic and therapeutic purposes set forth below.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, e.g., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The antibodies comprising an ultralong CDR3 as disclosed herein can bemade using a hybridoma cell-based method first described by Kohler etal., Nature, 256:495 (1975), or may be made by recombinant DNA methods.

Hybridoma cells can be generated by fusing B cells producing a desiredantibody with an immortalized cell line, usually a myeloma cell line, sothat the resulting fusion cells will be an immortalized cell line thatsecrets a particular antibody. By the same principle, myeloma cells canbe first transfected with a nucleic acid encoding a germline antibody Vregion and can be screened for the expression of the germline V region.Those myeloma cells with highest level of proteolytic light chainexpression can be subsequently fused with B cells that produce anantibody with desired target protein specificity. The fusion cells willproduce two types of antibodies: one is a heterologous antibodycontaining an endogenous antibody chain (either heavy or light) operablyjoined to the recombinant germline V region (either heavy or light), andthe other is the same antibody that the parental B cells would secrete(e.g. both endogenous heavy and light chains). The operably joinedheterologous heavy and light chains can be isolated by conventionalmethods such as chromatography and identification can be confirmed bytarget protein binding assays, assays identifying a unique tag of thegermline polypeptide, or endopeptidase activity assays described inother sections of this disclosure. In some cases, where the heterologousantibody is the predominant type in quantity among the two types ofantibodies, such isolation may not be needed.

The hybridoma cells may be seeded and grown in a suitable culture mediumthat preferably contains one or more substances that inhibit the growthor survival of the unfused, parental myeloma cells. For example, if theparental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,myeloma cell lines may be murine myeloma lines, such as those derivedfrom MOPC-21 and MPC-11 mouse tumors available from the Salk InstituteCell Distribution Center, San Diego, Calif. USA, and SP-2 or X⁶³-Ag8-653cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 (MarcelDekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of antibodies comprising an ultralong CDR3. For example, thebinding specificity of monoclonal antibodies produced by hybridoma cellsmay be determined by immunoprecipitation or by an in vitro bindingassay, such as an enzyme-linked immunoadsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

The antibodies comprising an ultralong CDR3 as disclosed herein may bemade by using combinatorial libraries to screen for synthetic antibodyclones with the desired activity or activities. For example, syntheticantibody clones are selected by screening phage libraries containingphage that display various fragments of antibody variable regions (e.g.,scFv or Fab) fused to phage coat protein. Such phage libraries may bepanned, for example, by affinity chromatography against the desiredantigen. Clones expressing antibody fragments capable of binding to thedesired antigen may be adsorbed to the antigen and thus separated fromthe non-binding clones in the library. The binding clones may then beeluted from the antigen, and can be further enriched by additionalcycles of antigen adsorption/elution. Any of the antibodies comprisingan ultralong CDR3 as disclosed herein may be obtained by designing asuitable antigen screening procedure to select for the phage clone ofinterest followed by construction of a full length antibody comprisingan ultralong CDR3 clone using the VH and VL (e.g., from scFv or Fab)sequences from the phage clone of interest and suitable constant region(Fc) sequences described in Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, NIH Publication 91-3242, BethesdaMd. (1991), vols. 1-3.

The antigen-binding domain of an antibody is formed from two variable(V) regions, one each from the light (VL) and heavy (VH) chains, thatboth present three hypervariable loops or complementarity-determiningregions (CDRs). Variable domains may be displayed functionally on phage,either as single-chain Fv (scFv, also referred to as single-chainantibody (SCA)) fragments, in which VH and VL are covalently linkedthrough a short, flexible peptide, or as Fab fragments, in which theyare each fused to a constant domain and interact non-covalently, asdescribed in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). scFvor SCA encoding phage clones and Fab encoding phage clones may beseparately or collectively referred to as “Fv phage clones” or “Fvclones”.

Repertoires of VH and VL genes may be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire may be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J. 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Filamentous phage is used to display antibody fragments by fusion to theminor coat protein pIII. Protein pIII may include truncated forms ofpIII. The antibody fragments can be displayed as single chain Fvfragments, in which VH and VL domains are connected on the samepolypeptide chain by a flexible polypeptide spacer, (e.g., as describedby Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or as Fabfragments, in which one chain is fused to pIII (e.g., a truncated pIII)and the other is secreted into the bacterial host cell periplasm whereassembly of a Fab-coat protein structure which becomes displayed on thephage surface by displacing some of the wild type coat proteins, (e.g.,as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137(1991)).

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and may beamplified or copies made by recombinant DNA techniques (e.g., Kunkelmutagenesis). For example, in the case of rearranged VH and VL genelibraries, the desired DNA may be obtained by isolating genomic DNA ormRNA from lymphocytes followed by polymerase chain reaction (PCR) withprimers matching the 5′ and 3′ ends of rearranged VH and VL genes asdescribed in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-3837(1989), thereby making diverse V gene repertoires for expression. The Vgenes may be amplified from cDNA and genomic DNA, with back primers atthe 5′ end of the exon encoding the mature V-domain and forward primersbased within the J-segment as described in Orlandi et al. (1989) and inWard et al., Nature, 341: 544-546 (1989). For amplifying from cDNA, backprimers can also be based in the leader exon as described in Jones etal., Biotechnol., 9: 88-89 (1991), and forward primers within theconstant region as described in Sastry et al., Proc. Natl. Acad. Sci.(USA), 86: 5728-5732 (1989). To enhance or maximize complementarity,degeneracy may be incorporated in the primers as described in Orlandi etal. (1989) or Sastry et al. (1989). Library diversity may be enhanced ormaximized by using PCR primers targeted to each V-gene family in orderto amplify available VH and VL arrangements present in the immune cellnucleic acid sample, for example, as described in the method of Marks etal., J. Mol. Biol., 222: 581-597 (1991) or as described in the method ofOrum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning ofthe amplified DNA into expression vectors, rare restriction may can beintroduced within the PCR primer as a tag at one end as described inOrlandi et al. (1989), or by further PCR amplification with a taggedprimer as described in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes may be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (e.g., reported in Tomlinson et al., J. Mol. Biol.,227: 776-798 (1992)), and mapped (e.g., reported in Matsuda et al.,Nature Genet., 3: 88-94 (1993); these cloned segments (including all themajor conformations of the H1 and H2 loop) may be used to generatediverse VH gene repertoires with PCR primers encoding H3 loops ofdiverse sequence and length as described in Hoogenboom and Winter, J.Mol. Biol., 227: 381-388 (1992). VH repertoires may also be made withall the sequence diversity focused in a long H3 loop of a single lengthas described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461(1992). Human Vκ and Vλ. segments have been cloned and sequenced(reported in Williams and Winter, Eur. J. Immunol., 23: 1456-1461(1993)) and can be used to make synthetic light chain repertoires.Synthetic V gene repertoires, based on a range of VH and VL folds, andL3 and H3 lengths, will encode antibodies of considerable structuraldiversity. Following amplification of V-gene encoding DNAs, germlineV-gene segments can be rearranged in vitro according to the methods ofHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Repertoires of antibody fragments may be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire may becreated in different vectors, and the vectors recombined in vitro, forexample, as described in Hogrefe et al., Gene, 128: 119-126 (1993), orin vivo by combinatorial infection, for example, the loxP systemdescribed in Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993).The in vivo recombination approach exploits the two-chain nature of Fabfragments to overcome the limit on library size imposed by E. colitransformation efficiency. Naive VH and VL repertoires are clonedseparately, one into a phagemid and the other into a phage vector. Thetwo libraries are then combined by phage infection ofphagemid-containing bacteria so that each cell contains a differentcombination and the library size is limited only by the number of cellspresent (about 10¹² clones). Both vectors contain in vivo recombinationsignals so that the VH and VL genes are recombined onto a singlereplicon and are co-packaged into phage virions. These large librariesmay provide large numbers of diverse antibodies of good affinity (K_(d)⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, for example, as described in Barbas et al., Proc. Natl. Acad.Sci. USA, 88: 7978-7982 (1991), or assembled together by PCR and thencloned, for example, as described in Clackson et al., Nature, 352:624-628 (1991). PCR assembly may also be used to join VH and VL DNAswith DNA encoding a flexible peptide spacer to form single chain Fv(scFv) repertoires. In yet another technique, “in cell PCR assembly” maybe used to combine VH and VL genes within lymphocytes by PCR and thenclone repertoires of linked genes as described in Embleton et al., Nucl.Acids Res., 20: 3831-3837 (1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷M⁻¹), butaffinity maturation may also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci. USA, 89: 3576-3580 (1992). Additionally, affinity maturationmay be performed by randomly mutating one or more CDRs, for example,using PCR with primers carrying random sequence spanning the CDR ofinterest, in selected individual Fv clones and screening for higheraffinity clones. WO 9607754 described a method for inducing mutagenesisin a complementarity determining region of an immunoglobulin light chainto create a library of light chain genes. Another effective approach isto recombine the VH or VL domains selected by phage display withrepertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10⁻⁹ M range.

The phage library samples are contacted with an immobilized proteinunder conditions suitable for binding of at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g., as described in Barbas et al., Proc.Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or by alkali, (e.g., asdescribed in Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or byantigen competition, (e.g., in a procedure similar to the antigencompetition method of Clackson et al., Nature, 352: 624-628 (1991)).Phages may be enriched 20-1,000-fold in a single round of selection.Moreover, the enriched phages may be grown in bacterial culture andsubjected to further rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)may be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) may bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

DNA encoding the hybridoma-derived monoclonal antibodies or phagedisplay Fv clones disclosed herein is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide primersdesigned to specifically amplify the heavy and light chain codingregions of interest from hybridoma or phage DNA template). Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis of thedesired monoclonal antibodies in the recombinant host cells. Recombinantexpression in bacteria of antibody-encoding DNA has been described byBetter et al., U.S. Pat. No. 6,204,023 (see also, e.g., Skerra et al.,Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs,130: 151 (1992)).

DNA encoding Fv clones as disclosed herein may be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g., the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions may be obtained from any humanor animal species. A Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid”, fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In a preferred Fv cloneembodiment, a Fv clone derived from human variable DNA is fused to humanconstant region DNA to form coding sequence(s) for all human, full orpartial length heavy and/or light chains.

DNA encoding an antibody comprising an ultralong CDR3 derived from ahybridoma disclosed herein may also be modified, for example, bysubstituting the coding sequence for human heavy- and light-chainconstant domains in place of homologous murine sequences derived fromthe hybridoma clone (e.g., as in the method of Morrison et al., Proc.Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNA encoding a hybridoma orFv clone-derived antibody or fragment can be further modified bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. In thismanner, “chimeric” or “hybrid” antibodies are prepared that have thebinding specificity of the Fv clone or hybridoma clone-derivedantibodies disclosed herein.

Antibody Genes and Proteins

The present disclosure provides antibody genes and proteins including,for example, chimeric, recombinant, engineered, synthetic, hybrid,bovine, fully bovine, bovinized, human, fully human or humanizedantibody genes or proteins that comprise an ultralong CDR3 sequence. Theantibodies disclosed herein may selectively or specifically bind to anepitope of a target protein. In some embodiments, the antibody may be anantagonist (e.g., blocking) antibody or an agonist antibody.

The variable region of the heavy and light chains are encoded bymultiple germline gene segments separated by non-coding regions, orintrons, and often are present on different chromosomes. For example,the genes for the human immunoglobulin heavy chain region containsapproximately 65 variable (VH) genes, 27 Diversity (DH) genes, and 6Joining (JH) genes. The human kappa (κ) and lambda (λ) light chains arealso each encoded by a similar number of VL and JL gene segments, but donot include any D gene segments. Exemplary VH, DH, JH and VL (Vκ or Vλ)and JL (Jκ or Jλ) germline gene segments are set forth in WO2010/054007.

During B cell differentiation germline DNA is rearranged whereby one DHand one JH gene segment of the heavy chain locus are recombined, whichis followed by the joining of one VH gene segment forming a rearrangedVDJ gene that encodes a VH chain. The rearrangement occurs only on asingle heavy chain allele by the process of allelic exclusion. Allelicexclusion is regulated by in-frame or “productive” recombination of theVDJ segments, which occurs in only about one-third of VDJ recombinationsof the variable heavy chain. When such productive recombination eventsfirst occur in a cell, this result in production of a μ heavy chain thatgets expressed on the surface of a pre-B cell and transmits a signal toshut off further heavy chain recombination, thereby preventingexpression of the allelic heavy chain locus. The surface-expressed μheavy chain also acts to activate the kappa (κ) locus for rearrangement.The lambda (λ) locus is only activated for rearrangement if the Krecombination is unproductive on both loci. The light chainrearrangement events are similar to the heavy chain, except that onlythe VL and JL segments are recombined. Before primary transcription ofeach, the corresponding constant chain gene is added. Subsequenttranscription and RNA splicing leads to mRNA that is translated into anintact light chain or heavy chain.

The variable regions of antibodies confer antigen binding andspecificity due to recombination events of individual germline V, D andJ segments, whereby the resulting recombined nucleic acid sequencesencoding the variable region domains differ among antibodies and conferantigen-specificity to a particular antibody. The variation, however, islimited to three complementarity determining regions (CDR1, CDR2, andCDR3) found within the N-terminal domain of the heavy (H) and (L) chainvariable regions. The CDRs are interspersed with regions that are moreconserved, termed “framework regions” (FR). The extent of the frameworkregion and CDRs has been precisely defined (see e.g., Kabat, E. A. etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917).Each VH and VL is typically composed of three CDRs and four FRs arrangedfrom the amino terminus to carboxy terminus in the following order: FR1,CDR1, FR2, CDR2, FR3, CDR3 and FR4. Sequence variability among VL and VHdomains is generally limited to the CDRs, which are the regions thatform the antigen binding site. For example, for the heavy chain,generally, VH genes encode the N-terminal three framework regions, thefirst two complete CDRs and the first part of the third CDR), the DHgene encodes the central portion of the third CDR, and the JH geneencodes the last part of the third CDR and the fourth framework region.For the light chain, the VL genes encode the first CDR and second CDR.The third CDR (CDRL3) is formed by the joining of the VL and JL genesegments. Hence, CDRs 1 and 2 are exclusively encoded by germline V genesegment sequences. The VH and VL chain CDR3s form the center of theAg-binding site, with CDRs 1 and 2 form the outside boundaries; the FRssupport the scaffold by orienting the H and L CDRs. On average, anantigen binding site typically requires at least four of the CDRs makecontact with the antigen's epitope, with CDR3 of both the heavy andlight chain being the most variable and contributing the mostspecificity to antigen binding (see, e.g., Janis Kuby, Immunology, ThirdEdition, New York, W.H. Freeman and Company, 1998, pp. 115-118). CDRH3,which includes all of the D gene segment, is the most diverse componentof the Ab-binding site, and typically plays a critical role in definingthe specificity of the Ab. In addition to sequence variation, there isvariation in the length of the CDRs between the heavy and light chains.

The constant regions, on the other hand, are encoded by sequences thatare more conserved among antibodies. These domains confer functionalproperties to antibodies, for example, the ability to interact withcells of the immune system and serum proteins in order to causeclearance of infectious agents. Different classes of antibodies, forexample IgM, IgD, IgG, IgE and IgA, have different constant regions,allowing them to serve distinct effector functions.

These natural recombination events of V, D, and J, can provide nearly2×10⁷ different antibodies with both high affinity and specificity.Additional diversity is introduced by nucleotide insertions anddeletions in the joining segments and also by somatic hypermutation of Vregions. The result is that there are approximately 10¹⁰ antibodiespresent in an individual with differing antigen specificities.

Antibody Fragments

The present disclosure encompasses antibody fragments. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors.Antibody fragments include, but are not limited to, Fab, Fab′, Fab′-SH,F(ab′)2, Fv, Fv′, Fd, Fd′, scFv, hsFv fragments, and diabodies, andother fragments described below. For a review of certain antibodyfragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review ofscFv fragments, see, e.g., Pluckthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, NewYork), pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458. For discussion of Fab and F(ab′)2 fragmentscomprising salvage receptor binding epitope residues and havingincreased in vivo half-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9: 129134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516). Antibodyfragments can be made by various techniques, including but not limitedto proteolytic digestion of an intact antibody as well as production byrecombinant host cells (e.g. E. coli or phage), as described herein.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments (see,e.g., U.S. Pat. No. 6,204,023). Antibody fragments can be isolated fromantibody phage libraries as discussed above. Alternatively, Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)₂ fragments (see, e.g., Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab′)₂ fragments can beisolated directly from recombinant host cell culture. Fab and F(ab′)₂fragment with increased in vivo half-life comprising a salvage receptorbinding epitope residues (see, e.g., in U.S. Pat. No. 5,869,046). Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner. In other embodiments, the antibody of choiceis a single chain Fv fragment (scFv or single chain antibody (SCA)). SeeWO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458. Fv and sFv are theonly species with intact combining sites that are devoid of constantregions; thus, they are suitable for reduced nonspecific binding duringin vivo use. sFv fusion proteins may be constructed to yield fusion ofan effector protein at either the amino or the carboxy terminus of ansFv. See Antibody Engineering, ed. Borrebaeck, Supra. The antibodyfragment may also be a “linear antibody”, for example, as described inU.S. Pat. No. 5,641,870. Such linear antibody fragments may bemonospecific or bispecific.

Humanized Antibodies

The present disclosure provides humanized antibodies comprising anultralong CDR3. Humanized antibodies may include human engineeredantibodies (see, e.g., Studnicka et al. (1994) Protein Eng. 7(6)805-814; and U.S. Pat. No. 5,766,886). Various methods for humanizingnon-human antibodies are known in the art. For example, a humanizedantibody can have one or more amino acid residues introduced into itfrom a source which is human or non-human. Humanization may be performedfollowing the method of Studnicka (see, e.g., Studnicka et al. (1994)Protein Eng. 7(6) 805-814; and U.S. Pat. No. 5,766,886), including thepreparation of modified antibody variable domains. Humanization mayalternatively be performed following the method of Winter and co-workers(Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), bysubstituting hypervariable region sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” or “humanengineered” antibodies are chimeric antibodies, including whereinsubstantially less than an intact human variable domain has beensubstituted by or incorporated into the corresponding sequence from anon-human species. For example, humanized antibodies may be humanantibodies in which some hypervariable region residues and possibly someFR residues are substituted by residues from analogous sites in rodentantibodies. Alternatively, humanized or human engineered antibodies maybe non-human (e.g, rodent) antibodies in which some residues aresubstituted by residues from analogious sites in human antibodies (see,e.g., Studnicka et al. (1994) Protein Eng. 7(6) 805-814; and U.S. Pat.No. 5,766,886).

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is important to reduce antigenicity.For example, to the so-called “best-fit” method, the sequence of thevariable domain of a rodent antibody is screened against the entirelibrary of known human variable-domain sequences. The human sequencewhich is closest to that of the rodent is then accepted as the humanframework for the humanized antibody (Sims et al. (1993) J. Immunol.151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901). Another methoduses a particular framework derived from the consensus sequence of allhuman antibodies of a particular subgroup of light or heavy chains. Thesame framework may be used for several different humanized antibodies(Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al.(1993) J. Immunol., 151:2623).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, e.g., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

In some embodiments, the humanized antibodies comprising an ultralongCDR3 may be deimmunized. Methods of deimmunizing an antibody or proteinare well known in the art. The immunogenicity of therapeutic proteinssuch as antibodies is thought to result from the presence of T-cellepitopes which can bind MHC class II molecules and generate aproliferative and cytokine response in CD4+ helper T-cells. These CD4+helper cells then collaborate with B-cells to generate an antibodyresponse against the therapeutic protein. Removal of the T-cell epitopesare thought to be key steps in deimmunizing a recombinant protein.T-cell epitopes can be predicted by in silico algorithms that identifyresidues required for binding MHC. Alternatively, epitopes can beidentified directly by utilizing peripheral blood mononuclear cells frompanels of human donors and measuring their response against thetherapeutic protein when incubated with antigen presenting cells. Suchin silico and in vitro systems are well known in the art [Jones T D,Crompton L J, Can F J, Baker M P. Methods Mol Biol. 2009; 525:405-23,Deimmunization of monoclonal antibodies; and Baker M, and Jones T D. Theidentification and removal of immunogenicity in therapeutic proteins.Curr. Opin. Drug Discovery Dev. 2007; (2007); 10(2): 219-227]. Whenpeptides are identified that bind MHC II or otherwise stimulate CD4+cell activation, the residues of the peptide can be mutated one by oneand tested for T-cell activation until a mutation is found whichdisrupts MHC II binding and T-cell activation. Such mutations, whenfound in an individual peptide, can be encoded directly in therecombinant therapeutic protein. Incubation of the whole protein withantigen presenting cells will not induce a significant CD4+ response,indicating successful deimmunization.

Bovine Antibodies

The present disclosure provides for bovine antibodies comprising anultralong CDR3. The bovine antibodies may be recombinant antibodies,engineered antibodies, synthetic antibodies, bovinized antibodies, orfully bovine antibodies. Bovinized antibodies may include bovineengineered antibodies. Methods for producing a bovinized antibody maycomprise introducing one or more amino acid residues into it from asource which is a bovine. In some instances, methods for producing abovinized antibody may comprise introducing one or more amino acidresidues into it from a source which is a non-bovine. Bovinization maybe performed by preparing a modified antibody variable domains.Alternatively, bovinization may be performed by substitutinghypervariable region sequences for the corresponding sequences of abovine antibody. Accordingly, such “bovinized” or “bovine engineered”antibodies are chimeric antibodies. Chimeric antibodies may includeantibodies wherein substantially less than an intact bovine variabledomain has been substituted by or incorporated into the correspondingsequence from a non-bovine species. Bovinized or bovine engineeredantibodies may be bovine antibodies in which some hypervariable regionresidues and constant region residues are substituted by residues fromanalogous sites in non-bovine antibodies. Alternatively, bovinized,bovine engineered or fully bovine antibodies may be non-bovine (e.g,human) antibodies in which some residues are substituted by residuesfrom analogious sites in bovine antibodies. For example, a bovineimmunoglobuline region can be used to replace a non-bovine (e.g., human,rodent) immunoglobulin region to produce a fully bovine, bovinized orbovine engineered antibody.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. For example, one of the binding specificities may be for afirst antigen and the other may be for any other antigen. Exemplarybispecific antibodies may bind to two different epitopes of the sameprotein. Bispecific antibodies may also be used to localize cytotoxicagents to cells which express a particular protein. These antibodiespossess a binding arm specific for the particular protein and an armwhich binds the cytotoxic agent (e.g., saporin, anti-interferon-α, vincaalkaloid, ricin A chain, methotrexate or radioactive isotope hapten).Bispecific antibodies may be prepared as full length antibodies orantibody fragments (e.g., F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10: 3655 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1), containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are not ofparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm.This asymmetric structure may facilitate the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. Thisapproach is disclosed in WO 94/04690. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules may can be engineered to maximize the percentage ofheterodimers which are recovered from recombinant cell culture. Thepreferred interface comprises at least a part of the C_(H3) domain of anantibody constant domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatemay be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies may be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced maybe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. See, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992). The leucine zipper peptides from the Fos andJun proteins were linked to the Fab′ portions of two differentantibodies by gene fusion. The antibody homodimers were reduced at thehinge region to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a heavy-chain variable domain (VH) connected to alight-chain variable domain (VL) by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. See, e.g., Tutt et al. J.Immunol. 147: 60 (1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present disclosure may bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g., tetravalent antibodies),which may be produced by recombinant expression of nucleic acid encodingthe polypeptide chains of the antibody. The multivalent antibody maycomprise a dimerization domain and three or more antigen binding sites.A preferred dimerization domain may comprise (or consist of) an Fcregion or a hinge region. In this scenario, the antibody will comprisean Fc region and three or more antigen binding sites amino-terminal tothe Fe region. A preferred multivalent antibody may comprise (or consistof) three to about eight, but preferably four, antigen binding sites.The multivalent antibody comprises at least one polypeptide chain (andpreferably two polypeptide chains), wherein the polypeptide chain(s)comprise two or more variable domains. For instance, the polypeptidechain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a firstvariable domain, VD2 is a second variable domain, Fc is one polypeptidechain of an Fc region, X1 and X2 represent an amino acid or polypeptide,and n is 0 or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. A multivalent antibody may preferably further comprises atleast two (and preferably four) light chain variable domainpolypeptides. A multivalent antibody may, for instance, comprise fromabout two to about eight light chain variable domain polypeptides. Thelight chain variable domain polypeptides may comprise a light chainvariable domain and, optionally, further comprise a CL domain.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies comprising an ultralong CDR3 as described herein arecontemplated. For example, it may be desirable to improve the bindingaffinity and/or other biological properties of the antibody. Amino acidsequence variants including, for example, conservatively modifiedvariants, of the antibody are prepared by introducing appropriatenucleotide changes into the antibody nucleic acid, or by peptidesynthesis. Such modifications include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid alterations may be introduced in the subject antibodyamino acid sequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g., for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody have been described (see, e.g., US 2003/0157108, US2004/0093621. Antibodies with a bisecting N-acetylglucosamine (GlcNAc)in the carbohydrate attached to an Fc region of the antibody have beendescribed (see, e.g., WO 2003/011878, and U.S. Pat. No. 6,602,684).Antibodies with at least one galactose residue in the oligosaccharideattached to an Fc region of the antibody WO 1997/30087; see, also, WO1998/58964 and WO 1999/22764 concerning antibodies with alteredcarbohydrate attached to the Fc region thereof). Antigen-bindingmolecules with modified glycosylation have been described (see, e.g., WO99/54342, U.S. Pat. Nos. 6,602,684 and 7,517,670, and US 2004/0072290;see also, e.g., U.S. Pat. Nos. 7,214,775 and 7,682,610).

The preferred glycosylation variant herein comprises an Fc region,wherein a carbohydrate structure attached to the Fc region lacks fucose.Such variants have improved ADCC function. Optionally, the Fc regionfurther comprises one or more amino acid substitutions therein whichfurther improve ADCC, for example, substitutions at positions 298, 333,and/or 334 of the Fc region (Eu numbering of residues). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodiesinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614(now U.S. Pat. No. 6,946,292) US 2002/0164328 (now U.S. Pat. No.7,064,191); US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282 (now U.S. Pat. No. 7,749,753); US 2004/0109865; WO2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778;WO2005/053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004);Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of celllines producing defucosylated antibodies include Lec13 CHO cellsdeficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys.249:533-545 (1986); US Pat Appl No US 2003/0157108 A1, Presta, L; and WO2004/056312 A1, Adams et al., especially at Example 11), and knockoutcell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockoutCHO cells (Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 2 under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions”, or asfurther described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring amino acids are divided into groups based on common side-chainproperties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2)neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: asp, glu; (4)basic: his, lys, arg; (5) residues that influence chain orientation:gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.,6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibodies thus generated are displayed fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and antigen. Such contact residuesand neighboring residues are candidates for substitution according tothe techniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides disclosed herein,thereby generating a Fc region variant. The Fc region variant maycomprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 orIgG4 Fc region) comprising an amino acid modification (e.g., asubstitution) at one or more amino acid positions including that of ahinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody used in methodsdisclosed herein may comprise one or more alterations as compared to thewild type counterpart antibody, e.g., in the Fc region. These antibodieswould nonetheless retain substantially the same characteristics requiredfor therapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (e.g., either improved ordiminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants. WO00/42072and WO 2004/056312 describe antibody variants with improved ordiminished binding to FcRs. See, also, Shields et al. J. Biol. Chem.9(2): 6591-6604 (2001). Antibodies with increased half lives andimproved binding to the neonatal Fc receptor (FcRn), which isresponsible for the transfer of maternal IgGs to the fetus (Guyer etal., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249(1994)), are described in US2005/0014934 (Hinton et al.). Theseantibodies comprise an Fc reg on with one or more substitutions thereinwhich improve binding of the Fc region to FcRn. Polypeptide variantswith altered Fc region amino acid sequences and increased or decreasedClq binding capability are described in U.S. Pat. No. 6,194,551,WO99/51642. See, also, Idusogie et al. J. Immunol. 164:4178-4184 (2000).

In certain embodiments, the present disclosure contemplates an antibodyvariant that possesses some but not all effector functions, which makeit a desirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'! Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see, Bruggemann, M. et al., Exp. Med. 166:1351-1361 (1987)).Alternatively, non-radioactive assays methods may be employed (see, forexample, ACTI™ non-radioactive cytotoxicity assay for flow cytometry(CellTecllrlology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq binding assays mayalso be carried out to confirm that the antibody is unable to bind Clqand hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al.,Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S, and M. J. Glennie, Blood103:27382743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) Clq binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. Immunol. 164: 41784184(2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., Immunol. 117:587 (1976) andKim et al., Immunol. 24:249 (1994)), are described in US2005/0014934A1(Hinton et al.). Those antibodies comprise an Fc region with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Such Fc variants include those with substitutions at one or more of Fcregion residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317,340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g.,substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

Antibody Derivatives

The antibodies comprising an ultralong CDR3 as disclosed herein may befurther modified to contain additional nonproteinaceous moieties thatare known in the art and readily available. Preferably, the moietiessuitable for derivatization of the antibody are water soluble polymers.Non-limiting examples of water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymers areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody or fragment thereof asdisclosed herein, the nucleic acid encoding it is isolated and insertedinto a replicable vector for further cloning (amplification of the DNA)or for expression. DNA encoding the antibody is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the antibody). In an exemplary embodiment,nucleic acid encoding an antibody comprising an ultralong CDR3, avariable region comprising an ultralong CDR3, or an ultralong CDR3, isisolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. Many vectors areavailable. The choice of vector depends in part on the host cell to beused. Generally, preferred host cells are of either prokaryotic oreukaryotic (generally mammalian) origin. It will be appreciated thatconstant regions of any isotype can be used for this purpose, includingIgG, IgM, IgA, IgD, and IgE constant regions, and that such constantregions can be obtained from any human or animal species.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the CMV promoter, SV40early promoter, SV40 later promoter, metallothionein promoter, murinemammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrinpromoter, or other promoters shown effective for expression ineukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect cells, with anucleic acid sequence encoding a partially human ultralong CDR3 antibodychain under the direction of the polyhedrin promoter or other strongbaculovirus promoters.

a. Generating Antibodies Using Prokaryotic or Eukaryotic Host Cells:

i. Vector Construction

Polynucleotide sequences encoding polypeptide components of theantibodies disclosed herein can be obtained using standard recombinanttechniques. Desired polynucleotide sequences may be isolated andsequenced from antibody producing cells such as hybridoma cells.Alternatively, polynucleotides can be synthesized using nucleotidesynthesizer or PCR techniques. Once obtained, sequences encoding thepolypeptides are inserted into a recombinant vector capable ofreplicating and expressing heterologous polynucleotides in prokaryotichosts. Many vectors that are available and known in the art can be usedfor the purpose of the present disclosure. Selection of an appropriatevector will depend mainly on the size of the nucleic acids to beinserted into the vector and the particular host cell to be transformedwith the vector. Each vector contains various components, depending onits function (amplification or expression of heterologouspolynucleotide, or both) and its compatibility with the particular hostcell in which it resides. The vector components generally include, butare not limited to: an origin of replication, a selection marker gene, apromoter, a ribosome binding site (RBS), a signal sequence, theheterologous nucleic acid insert and a transcription terminationsequence. Additionally, V regions comprising an ultralong CDR3 mayoptionally be fused to a C-region to produce an antibody comprisingconstant regions.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies have been described (see, e.g.,U.S. Pat. No. 5,648,237).

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vectors disclosed herein may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g., the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector disclosed herein. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include: an ara Bpromoter, a PhoA promoter, β-galactamase and lactose promoter systems, atryptophan (trp) promoter system and hybrid promoters such as the tac orthe trc promoter. However, other promoters that are functional inbacteria (such as other known bacterial or phage promoters) are suitableas well. Their nucleotide sequences have been published, therebyenabling a skilled worker operably to ligate them to cistrons encodingthe target light and heavy chains (e.g., Siebenlist et al. (1980) Cell20: 269) using linkers or adaptors to supply any required restrictionsites.

Suitable bacterial promoters are well known in the art and fullydescribed in scientific literature such as Sambrook and Russell, supra,and Ausubel et al, supra. Bacterial expression systems for expressingantibody chains of the recombinant catalytic polypeptide are availablein, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene,22:229-235 (1983); Mosbach et al., Nature, 302:543-545 (1983)).

In one aspect disclosed herein, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence should be one that is recognized andprocessed (e.g., cleaved by a signal peptidase) by the host cell. Forprokaryotic host cells that do not recognize and process the signalsequences native to the heterologous polypeptides, the signal sequenceis substituted by a prokaryotic signal sequence selected, for examplePelB, OmpA, alkaline phosphatase, penicillinase, Ipp, or heat-stableenterotoxin II (STII) leaders, LamB, PhoE, and MBP. In one embodimentdisclosed herein, the signal sequences used in both cistrons of theexpression system are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe disclosure can occur in the cytoplasm of the host cell, andtherefore does not require the presence of secretion signal sequenceswithin each cistron. In that regard, immunoglobulin light and heavychains are expressed, folded and assembled to form functionalimmunoglobulins within the cytoplasm. Certain host strains (e.g., the E.coli trxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits (see e.g., Proba and Pluckthun Gene,159:203 (1995)).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. In oneembodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary(CHO) cell, Human Embryonic Kidney (HEK) cell or lymphoid cell (e.g.,YO, NSO, Sp20 cell). For example, antibodies may be produced inbacteria, in particular when glycosylation and Fc effector function arenot needed. For expression of antibody fragments and polypeptides inbacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523.(See also Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo,ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describingexpression of antibody fragments in E. coli.) After expression, theantibody may be isolated from the bacterial cell paste in a solublefraction and can be further purified. In addition to prokaryotes,eukaryotic microbes such as filamentous fungi or yeast are suitablecloning or expression hosts for antibody-encoding vectors, includingfungi and yeast strains whose glycosylation pathways have been“humanized,” resulting in the production of an antibody with a partiallyor fully human glycosylation pattern. See Gemgross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. These examples are illustrative rather thanlimiting. Methods for constructing derivatives of any of theabove-mentioned bacteria having defined genotypes are known in the artand described in, for example, Bass et al., Proteins, 8:309-314 (1990).It is generally necessary to select the appropriate bacteria taking intoconsideration replicability of the replicon in the cells of a bacterium.For example, E. coli, Serratia, or Salmonella species can be suitablyused as the host when well known plasmids such as pBR322, pBR325,pACYC177, or pKN410 are used to supply the replicon. Typically the hostcell should secrete minimal amounts of proteolytic enzymes, andadditional protease inhibitors may desirably be incorporated in the cellculture.

Plant cell cultures can also be utilized as hosts. See, e.g. U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125, 978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants). Vertebrate cells may also be used as hosts. Forexample, mammalian cell lines that are adapted to grow in suspension maybe useful. Other examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line(293 or 293 cells as described, e.g., in Graham et al., Gen VII′01.36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980));monkey kidney cells (CV1); African green monkey kidney cells (V ERO-76);human cervical carcinoma cells (HELA); canine kidney cells (MDCK;buffalo rat liver cells (BRL 3A); human lung cells (W138); human livercells (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells, asdescribed, e.g., in Mather et al., Annals NI'. Acad. Sci. 383:44-68(1982); MRC 5 cells; and FS4 cells. Other useful mammalian host celllines include Chinese hamster ovary (CHO) cells, including DHFR′ CHOcells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); andmyeloma cell lines such as YO, NSO and Sp2/0. For a review of certainmammalian host cell lines suitable for antibody production, see, e.g.,Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,Humana Press, Totowa, N.].), pp. 255-268 (2003).

In one such embodiment, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.

ii. Antibody Production

For recombinant production of a partially human ultralong CDR3 antibody,nucleic acid encoding an antibody comprising an ultralong CDR3 isinserted into one or more expression vectors for further cloning and/orexpression in a host cell. Such nucleic acid may be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the antibody). Host cells are transformed withsuch expression vectors and cultured in conventional nutrient mediamodified as appropriate for inducing promoters, selecting transformants,or amplifying the genes encoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides disclosed herein aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector disclosedherein, protein expression is induced under conditions suitable for theactivation of the promoter. For example, an ara B or phoA promoter maybe used for controlling transcription of the polypeptides. A variety ofinducers may be used, according to the vector construct employed, as isknown in the art.

The expressed polypeptides of the present disclosure are secreted intoand recovered from the periplasm of the host cells or transported intothe culture media. Protein recovery from the periplasm typicallyinvolves disrupting the microorganism, generally by such means asosmotic shock, sonication or lysis. Once cells are disrupted, celldebris or whole cells may be removed by centrifugation or filtration.The proteins may be further purified, for example, by affinity resinchromatography. Alternatively, proteins that are transported into theculture media may be isolated therein. Cells may be removed from theculture and the culture supernatant being filtered and concentrated forfurther purification of the proteins produced. The expressedpolypeptides can be further isolated and identified using commonly knownmethods such as polyacrylamide gel electrophoresis (PAGE) and Westernblot assay.

Antibody production may be conducted in large quantity by a fermentationprocess. Various large-scale fed-batch fermentation procedures areavailable for production of recombinant proteins. Large-scalefermentations have at least 1000 liters of capacity, preferably about1,000 to 100,000 liters of capacity. These fermentors use agitatorimpellers to distribute oxygen and nutrients, especially glucose (apreferred carbon/energy source). Small scale fermentation refersgenerally to fermentation in a fermentor that is no more thanapproximately 100 liters in volumetric capacity, and can range fromabout 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptidesdisclosed herein, various fermentation conditions can be modified. Forexample, to improve the proper assembly and folding of the secretedantibody polypeptides, additional vectors overexpressing chaperoneproteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) orFkpA (a peptidylprolyl cis, trans-isomerase with chaperone activity) maybe used to co-transform the host prokaryotic cells. The chaperoneproteins have been demonstrated to facilitate the proper folding andsolubility of heterologous proteins produced in bacterial host cells.(see e.g., Chen et al. (1999) J Bio Chem 274:19601-19605; U.S. Pat. No.6,083,715; U.S. Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J.Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem.275:17106-17113; Arie et al. (2001) Mol. Microbiol. 39:199-210).

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the presentdisclosure. For example, host cell strains may be modified to effectgenetic mutation(s) in the genes encoding known bacterial proteases suchas Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available (see, e.g., Joly et al. (1998), supra; U.S. Pat.No. 5,264,365; U.S. Pat. No. 5,508,192; Hara et al., Microbial DrugResistance, 2:63-72 (1996)).

E. coli strains deficient for proteolytic enzymes and transformed withplasmids overexpressing one or more chaperone proteins may be used ashost cells in the expression systems disclosed herein.

iii. Antibody Purification

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, and gel filtration using, for example,Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody productsdisclosed herein. Protein A is a 41 kD cell wall protein fromStaphylococcus aureas which binds with a high affinity to the Fc regionof antibodies (see, e.g., Lindmark et al (1983) J. Immunol. Meth.62:1-13). The solid phase to which Protein A is immobilized ispreferably a column comprising a glass or silica surface, morepreferably a controlled pore glass column or a silicic acid column. Insome applications, the column has been coated with a reagent, such asglycerol, in an attempt to prevent nonspecific adherence ofcontaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

b. Generating Antibodies Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (e.g., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may be usedonly because it contains the early promoter.

(Iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. Alternatively, the Rous Sarcoma Virus long terminal repeatcan be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of thisdisclosure by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein, andinsulin). An enhancer from a eukaryotic cell virus may also be used.Examples include the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) onenhancing elements for activation of eukaryotic promoters. The enhancermay be spliced into the vector at a position 5′ or 3′ to the antibodypolypeptide-encoding sequence, but is preferably located at a site 5′from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,biolistics, liposomes, microinjection, plasma vectors, viral vectors andany of the other well known methods for introducing cloned genomic DNA,cDNA, synthetic DNA, or other foreign genetic material into a host cell(see, e.g., Sambrook and Russell, supra). It is only necessary that theparticular genetic engineering procedure used be capable of successfullyintroducing at least both genes into the host cell capable of expressinggermline antibody polypeptide.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this disclosure may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. PatentReissue 30,985 may be used as culture media for the host cells. Any ofthese media may be supplemented as necessary with hormones and/or othergrowth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon® ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Soluble forms of antibody or fragment present either in the cytoplasm orreleased from the periplasmic space may be further purified usingmethods known in the art, for example Fab fragments are released fromthe bacterial periplasmic space by osmotic shock techniques.

If inclusion bodies comprising an antibody or fragment have formed, theycan often bind to the inner and/or outer cellular membranes and thuswill be found primarily in the pellet material after centrifugation. Thepellet material can then be treated at pH extremes or with chaotropicagent such as a detergent, guanidine, guanidine derivatives, urea, orurea derivatives in the presence of a reducing agent such asdithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pHto release, break apart, and solubilize the inclusion bodies. Thesoluble antibody or fragment can then be analyzed using gelelectrophoresis, immunoprecipitation or the like. If it is desired toisolate a solublized antibody or antigen binding fragment isolation maybe accomplished using standard methods such as those set forth below andin Marston et al. (Meth. Enz., 182:264-275 (1990)).

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25 M salt).

In some cases, an antibody or fragment may not be biologically activeupon isolation. Various methods for “refolding” or converting apolypeptide to its tertiary structure and generating disulfide linkages,can be used to restore biological activity. Such methods includeexposing the solubilized polypeptide to a pH usually above 7 and in thepresence of a particular concentration of a chaotrope. The selection ofchaotrope is very similar to the choices used for inclusion bodysolubilization, but usually the chaotrope is used at a lowerconcentration and is not necessarily the same as chaotropes used for thesolubilization. In most cases the refolding/oxidation solution will alsocontain a reducing agent or the reducing agent plus its oxidized form ina specific ratio to generate a particular redox potential allowing fordisulfide shuffling to occur in the formation of the protein's cysteinebridge(s). Some of the commonly used redox couples includecysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, and2-mercaptoethanol(bME)/di-thio-b(ME). In many instances, a cosolvent maybe used to increase the efficiency of the refolding, and common reagentsused for this purpose include glycerol, polyethylene glycol of variousmolecular weights, arginine and the like.

Immunoconjugates

The disclosure also provides immunoconjugates (interchangeably termed“antibody-drug conjugates” or “ADC”), comprising any of the antibodiescomprising an ultralong CDR3 as described herein conjugated to acytotoxic agent such as a chemotherapeutic agent, a drug, a growthinhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (e.g., a radioconjugate). Alternatively, theimmunoconjugate comprises any of the antibodies comprising an ultralongCDR3 as described herein conjugated to a peptide. The peptide may be anon-antibody peptide, therapeutic polypeptide, cytokine, hormone orgrowth factor. The peptide may be encoded by a non-antibody sequence.

The antibody-drug conjugates may be used for the local delivery ofcytotoxic or cytostatic agents. For example, drugs to kill or inhibittumor cells in the treatment of cancer (Syrigos and Epenetos (1999)Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv.Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) allows targeteddelivery of the drug moiety to tumors, and intracellular accumulationtherein, where systemic administration of these unconjugated drug agentsmay result in unacceptable levels of toxicity to normal cells as well asthe tumor cells sought to be eliminated (Baldwin et al., (1986) Lancetpp. (Mar. 15, 1986):603-05; Thorpe, (1985) “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies'84: Biological And Clinical Applications, A. Pinchera et al. (ed.s),pp. 475-506). Maximal efficacy with minimal toxicity is sought thereby.Both polyclonal antibodies and monoclonal antibodies have been reportedas useful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) Supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Thetoxins may effect their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784)and are under therapeutic development.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein. Enzymatically active toxins and fragments thereof thatcan be used include diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. See, e.g., WO 93/21232 published Oct.28, 1993. A variety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin maybe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC 1065, and the derivatives of these toxins thathave toxin activity, are also contemplated herein.

a. Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) comprising an ultralong CDR3 as disclosed hereinconjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and EP 0 425 235. Liu et al., Proc. Natl. Acad.Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising amaytansinoid designated DM1 linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al.,Cancer Research 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansinoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10⁵ HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020. An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. Nos. 5,208,020, 6,441,163, or EP Patent 0 425235, Chari et al., Cancer Research 52:127-131 (1992).Antibody-maytansinoid conjugates comprising the linker component SMCCmay be prepared. The linking groups include disulfide groups, thioethergroups, acid labile groups, photolabile groups, peptidase labile groups,or esterase labile groups, as disclosed in the above-identified patents,disulfide and thioether groups being preferred. Additional linkinggroups are described and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson etal., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

b. Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibody disclosedherein conjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, (see, e.g., U.S. Pat. No.7,498,298).

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see, e.g., E. Schroder and K. Lubke, “ThePeptides”, volume 1, pp 76-136, 1965, Academic Press) that is well knownin the field of peptide chemistry. The auristatin/dolastatin drugmoieties may be prepared according to the methods of: U.S. Pat. No.5,635,483; U.S. Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem.Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725; and Pettitet al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863. See also Doronina(2003) Nat Biotechnol 21(7):778-784; U.S. Pat. No. 7,498,289,(disclosing, linkers and methods of preparing monomethylvaline compoundssuch as MMAE and MMAF conjugated to linkers).

c. Calicheamicin

In other embodiments, the immunoconjugate comprises an antibodydisclosed herein conjugated to one or more calicheamicin molecules. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296. Structural analogues of calicheamicin which may beused include, but are not limited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I),N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (see, e.g., Hinman et al., CancerResearch 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928(1998) and the aforementioned U.S. patents). Another anti-tumor drugthat the antibody can be conjugated is QFA which is an antifolate. Bothcalicheamicin and QFA have intracellular sites of action and do notreadily cross the plasma membrane. Therefore, cellular uptake of theseagents through antibody mediated internalization greatly enhances theircytotoxic effects.

d. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodiesdisclosed herein include BCNU, streptozoicin, vincristine and5-fluorouracil, the family of agents known collectively LL-E33288complex described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well asesperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present disclosure further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radiolabels or other labels may be incorporated in the conjugate inknown ways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds disclosed herein expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

e. Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC) disclosed herein, an antibody (Ab)is conjugated to one or more drug moieties (D), e.g., about 1 to about20 drug moieties per antibody, through a linker (L). An ADC of Formula I[Ab-(L-D)_(p)] may be prepared by several routes, employing organicchemistry reactions, conditions, and reagents known to those skilled inthe art, including: (1) reaction of a nucleophilic group of an antibodywith a bivalent linker reagent, to form Ab-L, via a covalent bond,followed by reaction with a drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a bivalent linker reagent, toform D-L, via a covalent bond, followed by reaction with thenucleophilic group of an antibody. Additional methods for preparing ADCare described herein.

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio)pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl (4-iodo-acetyl)aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are disclosed herein (see,e.g., U.S. Pat. No. 7,498,298).

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acids including analogs, such as citrulline. Amino acidlinker components can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g., lysine,(iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxylor amino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, e.g.,cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates disclosed herein may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g., withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g., by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either glactose oxidase or sodium meta-periodate may yieldcarbonyl (aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Engineered Hybridomas

Hybridoma cells can be generated by fusing B cells producing a desiredantibody with an immortalized cell line, usually a myeloma cell line, sothat the resulting fusion cells will be an immortalized cell line thatsecrets a particular antibody. By the same principle, myeloma cells canbe first transfected with a nucleic acid encoding a germline antibody Vregion and can be screened for the expression of the germline V region.Those myeloma cells with highest level of proteolytic light chainexpression can be subsequently fused with B cells that produce anantibody with desired target protein specificity. The fusion cells willproduce two types of antibodies: one is a heterologous antibodycontaining an endogenous antibody chain (either heavy or light) operablyjoined to the recombinant germline V region (either heavy or light), andthe other is the same antibody that the parental B cells would secrete(e.g. both endogenous heavy and light chains). The operably joinedheterologous heavy and light chains can be isolated by conventionalmethods such as chromatography and identification can be confirmed bytarget protein binding assays, assays identifying a unique tag of thegermline polypeptide, or endopeptidase activity assays described inother sections of this disclosure. In some cases, where the heterologousantibody is the predominant type in quantity among the two types ofantibodies, such isolation may not be needed. Hybridomas. Includingbovine hybridomas, may be a source of bovine antibody gene sequences,including ultralong CDR3 sequences.

Transgenic Mammals

A nucleic acid sequence encoding a germline antibody polypeptide of thepresent disclosure can be introduced into a non-human mammal to generatea transgenic animal that expresses the germline antibody polypeptide.Unlike the transgenic animal models more commonly seen, the transgeneexpressed by the transgenic mammals of the present disclosure need notreplace at least one allele of the endogenous coding sequenceresponsible for the variable regions of antibody chains followingsomatic recombination. Due to allelic exclusion, the presence of anexogenous, post-somatic rearrangement version of the germline V regionDNA will inhibit the endogenous alleles of pre-somatic rearrangement Vminigenes from undergoing somatic rearrangement and contributing to themakeup of antibody chains this mammal may produce. Thus, when exposed toa particular antigen, the mammal will generate heterologous antibodiescomprising one endogenously rearranged antibody chain, and onetransgenic gene which was rearranged a priori. Such heterologousantibodies are invaluable in research and in treating certain conditionsin live subjects. On the other hand, a method that directs theintegration of the transgene to the locus of an endogenous allele willfully serve the purpose of practicing the present disclosure as well.

The general methods of generating transgenic animals have been wellestablished and frequently practiced. For reviews and protocols forgenerating transgenic animals and related methods for geneticmanipulations, see, e.g., Mansour et al., Nature 336:348-352 (1988);Capecchi et al., Trends Genet. 5:70-76 (1989); Capecchi, Science244:1288-1292 (1989); Capecchi et al., Current Communications inMolecular Biology, pp 45-52, Capecchi, M. R. (ed.), Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989); Frohman et al., Cell 56: 145-147(1989); Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 (1985);Evans et. al., Nature 292:154-156 (1981); Bradley et al., Nature309:255-258 (1984); Gossler et al., Proc. Natl. Acad. Sci. USA83:9065-9069 (1986); Robertson et al., Nature 322:445-448 (1986);Jaenisch Science 240:1468-1474 (1988); and Siedel, G. E., Jr., “Criticalreview of embryo transfer procedures with cattle” in Fertilization andEmbryonic Development in Vitro, page 323, L. Mastroianni, Jr. and J. D.Biggers, ed., Plenum Press, New York, N.Y. (1981).

An exemplary transgenic animal of the present disclosure is mouse,whereas a number of other transgenic animals can also be produced usingthe same general method. These animals include, but are not limited to:rabbits, sheep, cattle, and pigs (Jaenisch Science 240:1468-1474 (1988);Hammer et al., J. Animal. Sci. 63:269 (1986); Hammer et al. Nature315:680 (1985); Wagner et al., Theriogenology 21:29 (1984)).

Pharmaceutical Compositions

Antibodies comprising an ultralong CDR3, antibody fragments, nucleicacids, or vectors disclosed herein can be formulated in compositions,especially pharmaceutical compositions. Such compositions withantibodies comprising an ultralong CDR3 comprise a therapeutically orprophylactically effective amount of antibodies comprising an ultralongCDR3, antibody fragment, nucleic acid, or vector disclosed herein inadmixture with a suitable carrier, e.g., a pharmaceutically acceptableagent. Typically, antibodies comprising an ultralong CDR3, antibodyfragments, nucleic acids, or vectors disclosed herein are sufficientlypurified for administration before formulation in a pharmaceuticalcomposition.

Pharmaceutically acceptable salts, excipients, or vehicles for use inthe present pharmaceutical compositions include carriers, excipients,diluents, antioxidants, preservatives, coloring, flavoring and dilutingagents, emulsifying agents, suspending agents, solvents, fillers,bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents,wetting agents, complexing agents, buffering agents, antimicrobials, andsurfactants.

Neutral buffered saline or saline mixed with serum albumin are exemplaryappropriate carriers. The pharmaceutical compositions may includeantioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, pluronics, or polyethylene glycol (PEG). Alsoby way of example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol, and the like. Suitable preservatives include benzalkoniumchloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid and the like. Hydrogen peroxide also may beused as preservative. Suitable cosolvents include glycerin, propyleneglycol, and PEG. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal, and the like. Thebuffers may be conventional buffers such as acetate, borate, citrate,phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH4-5.5, and Tris buffer can be about pH 7-8.5. Additional pharmaceuticalagents are set forth in Remington's Pharmaceutical Sciences, 18thEdition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

The composition may be in liquid form or in a lyophilized orfreeze-dried form and may include one or more lyoprotectants,excipients, surfactants, high molecular weight structural additivesand/or bulking agents (see, for example, U.S. Pat. Nos. 6,685,940,6,566,329, and 6,372,716). In one embodiment, a lyoprotectant isincluded, which is a non-reducing sugar such as sucrose, lactose ortrehalose. The amount of lyoprotectant generally included is such that,upon reconstitution, the resulting formulation will be isotonic,although hypertonic or slightly hypotonic formulations also may besuitable. In addition, the amount of lyoprotectant should be sufficientto prevent an unacceptable amount of degradation and/or aggregation ofthe protein upon lyophilization. Exemplary lyoprotectant concentrationsfor sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilizedformulation are from about 10 mM to about 400 mM. In another embodiment,a surfactant is included, such as for example, nonionic surfactants andionic surfactants such as polysorbates (e.g., polysorbate 20,polysorbate 80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol)phenyl ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodiumlaurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-,or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™. series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., Pluronics, PF68 etc). Exemplaryamounts of surfactant that may be present in the pre-lyophilizedformulation are from about 0.001-0.5%. High molecular weight structuraladditives (e.g., fillers, binders) may include for example, acacia,albumin, alginic acid, calcium phosphate (dibasic), cellulose,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,dextrin, dextrates, sucrose, tylose, pregelatinized starch, calciumsulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose,disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite,polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose,compressible sugar, magnesium aluminum silicate, maltodextrin,polyethylene oxide, polymethacrylates, povidone, sodium alginate,tragacanth microcrystalline cellulose, starch, and zein. Exemplaryconcentrations of high molecular weight structural additives are from0.1% to 10% by weight. In other embodiments, a bulking agent (e.g.,mannitol, glycine) may be included.

Compositions may be suitable for parenteral administration. Exemplarycompositions are suitable for injection or infusion into an animal byany route available to the skilled worker, such as intraarticular,subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral(intraparenchymal), intracerebroventricular, intramuscular, intraocular,intraarterial, or intralesional routes. A parenteral formulationtypically will be a sterile, pyrogen-free, isotonic aqueous solution,optionally containing pharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringers'dextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, anti-microbials, anti-oxidants, chelating agents, inertgases and the like. See generally, Remington's Pharmaceutical Science,16th Ed., Mack Eds., 1980.

Pharmaceutical compositions described herein may be formulated forcontrolled or sustained delivery in a manner that provides localconcentration of the product (e.g., bolus, depot effect) and/orincreased stability or half-life in a particular local environment. Thecompositions can include the formulation of antibodies comprising anultralong CDR3, antibody fragments, nucleic acids, or vectors disclosedherein with particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc., as well as agents such as abiodegradable matrix, injectable microspheres, microcapsular particles,microcapsules, bioerodible particles beads, liposomes, and implantabledelivery devices that provide for the controlled or sustained release ofthe active agent which then can be delivered as a depot injection.Techniques for formulating such sustained- or controlled-delivery meansare known and a variety of polymers have been developed and used for thecontrolled release and delivery of drugs. Such polymers are typicallybiodegradable and biocompatible. Polymer hydrogels, including thoseformed by complexation of enantiomeric polymer or polypeptide segments,and hydrogels with temperature or pH sensitive properties, may bedesirable for providing drug depot effect because of the mild andaqueous conditions involved in trapping bioactive protein agents (e.g.,antibodies comprising an ultralong CDR3). See, for example, thedescription of controlled release porous polymeric microparticles forthe delivery of pharmaceutical compositions in WO 93/15722.

Suitable materials for this purpose include polylactides (see, e.g.,U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids),such as poly-D-(−)-3-hydroxybutyric acid (EP 133,988A), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al.,J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyricacid. Other biodegradable polymers include poly(lactones),poly(acetals), poly(orthoesters), and poly(orthocarbonates).Sustained-release compositions also may include liposomes, which can beprepared by any of several methods known in the art (see, e.g., Eppsteinet al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrieritself, or its degradation products, should be nontoxic in the targettissue and should not further aggravate the condition. This can bedetermined by routine screening in animal models of the target disorderor, if such models are unavailable, in normal animals.

Microencapsulation of recombinant proteins for sustained release hasbeen performed successfully with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology. 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010. The sustained-release formulations of these proteins weredeveloped using poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be depending on its molecular weight and composition. Lewis,“Controlled release of bioactive agents from lactide/glycolide polymer,”in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as DrugDelivery Systems (Marcel Dekker: New York, 1990), pp. 1-41. Additionalexamples of sustained release compositions include, for example, EP58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No.1176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al.,Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. Release 90, 261[2003], Zhu et al., Nat. Biotechnol. 18, 24 [2000], and Dai et al.,Colloids Surf B Biointerfaces 41, 117 [2005].

Bioadhesive polymers are also contemplated for use in or withcompositions of the present disclosure. Bioadhesives are synthetic andnaturally occurring materials able to adhere to biological substratesfor extended time periods. For example, Carbopol and polycarbophil areboth synthetic cross-linked derivatives of poly(acrylic acid).Bioadhesive delivery systems based on naturally occurring substancesinclude for example hyaluronic acid, also known as hyaluronan.Hyaluronic acid is a naturally occurring mucopolysaccharide consistingof residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acidis found in the extracellular tissue matrix of vertebrates, including inconnective tissues, as well as in synovial fluid and in the vitreous andaqueous humor of the eye. Esterified derivatives of hyaluronic acid havebeen used to produce microspheres for use in delivery that arebiocompatible and biodegradable (see, for example, Cortivo et al.,Biomaterials (1991) 12:727-730; EP 517,565; WO 96/29998; Illum et al.,J. Controlled Rel. (1994) 29:133-141). Exemplary hyaluronic acidcontaining compositions of the present disclosure comprise a hyaluronicacid ester polymer in an amount of approximately 0.1% to about 40% (w/w)of an antibody comprising an ultralong CDR3 to hyaluronic acid polymer.

Both biodegradable and non-biodegradable polymeric matrices may be usedto deliver compositions of the present disclosure, and such polymericmatrices may comprise natural or synthetic polymers. Biodegradablematrices are preferred. The period of time over which release occurs isbased on selection of the polymer. Typically, release over a periodranging from between a few hours and three to twelve months is mostdesirable. Exemplary synthetic polymers which may be used to form thebiodegradable delivery system include: polymers of lactic acid andglycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkyleneglycols, polyalkylene oxides, polyalkylene terepthalates, polyvinylalcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides,polyurethanes and co-polymers thereof, poly(butic acid), poly(valericacid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers,cellulose esters, nitro celluloses, polymers of acrylic and methacrylicesters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxylethyl cellulose, cellulose triacetate,cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyethylene, polypropylene, poly(ethylene glycol),poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), polyvinyl acetate, poly vinyl chloride, polystyrene andpolyvinylpyrrolidone. Exemplary natural polymers include alginate andother polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion. The polymer optionally is in the formof a hydrogel (see, for example, WO 04/009664, WO 05/087201, Sawhney, etal., Macromolecules, 1993, 26, 581-587) that can absorb up to about 90%of its weight in water and further, optionally is cross-linked withmulti-valent ions or other polymers.

Delivery systems also include non-polymer systems that are lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the product is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in whicha product permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.Liposomes containing the product may be prepared by methods knownmethods, such as for example (DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; JP 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324).

Alternatively or additionally, the compositions may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which an antibody comprising anultralong CDR3, antibody fragment, nucleic acid, or vector disclosedherein has been absorbed or encapsulated. Where an implantation deviceis used, the device may be implanted into any suitable tissue or organ,and delivery of an antibody comprising an ultralong CDR3 antibodyfragment, nucleic acid, or vector disclosed herein can be directlythrough the device via bolus, or via continuous administration, or viacatheter using continuous infusion.

A pharmaceutical composition comprising an antibody comprising anultralong CDR3, antibody fragment, nucleic acid, or vector disclosedherein may be formulated for inhalation, such as for example, as a drypowder Inhalation solutions also may be formulated in a liquefiedpropellant for aerosol delivery. In yet another formulation, solutionsmay be nebulized. Additional pharmaceutical composition for pulmonaryadministration include, those described, for example, in WO 94/20069,which discloses pulmonary delivery of chemically modified proteins. Forpulmonary delivery, the particle size should be suitable for delivery tothe distal lung. For example, the particle size may be from 1 μm to 5μm; however, larger particles may be used, for example, if each particleis fairly porous.

Certain formulations containing antibodies comprising an ultralong CDR3,antibody fragments, nucleic acids, or vectors disclosed herein may beadministered orally. Formulations administered in this fashion may beformulated with or without those carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Forexample, a capsule can be designed to release the active portion of theformulation at the point in the gastrointestinal tract whenbioavailability is maximized and pre-systemic degradation is minimized.Additional agents may be included to facilitate absorption of aselective binding agent. Diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders also can be employed.

Another preparation may involve an effective quantity of an antibodycomprising an ultralong CDR3, antibody fragment, nucleic acid, or vectordisclosed herein in a mixture with non-toxic excipients which aresuitable for the manufacture of tablets. By dissolving the tablets insterile water, or another appropriate vehicle, solutions may be preparedin unit dose form. Suitable excipients include, but are not limited to,inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Suitable and/or preferred pharmaceutical formulations may be determinedin view of the present disclosure and general knowledge of formulationtechnology, depending upon the intended route of administration,delivery format, and desired dosage. Regardless of the manner ofadministration, an effective dose may be calculated according to patientbody weight, body surface area, or organ size. Further refinement of thecalculations for determining the appropriate dosage for treatmentinvolving each of the formulations described herein are routinely madein the art and is within the ambit of tasks routinely performed in theart. Appropriate dosages may be ascertained through use of appropriatedose-response data.

In some embodiments, antibodies comprising an ultralong CDR3 orfragments thereof are provided with a modified Fc region where anaturally-occurring Fc region is modified to increase the half-life ofthe antibody or fragment in a biological environment, for example, theserum half-life or a half-life measured by an in vitro assay. Methodsfor altering the original form of a Fc region of an IgG also aredescribed in U.S. Pat. No. 6,998,253.

In certain embodiments, it may be desirable to modify the antibody orfragment in order to increase its serum half-life, for example, addingmolecules such as PEG or other water soluble polymers, includingpolysaccharide polymers, to antibody fragments to increase thehalf-life. This may also be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis) (see, International Publication No. WO96/32478).Salvage receptor binding epitope refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule.

A salvage receptor binding epitope may include a region wherein any oneor more amino acid residues from one or two loops of a Fc domain aretransferred to an analogous position of the antibody fragment. Even morepreferably, three or more residues from one or two loops of the Fcdomain are transferred. Still more preferred, the epitope is taken fromthe CH2 domain of the Fc region (e.g., of an IgG) and transferred to theCH1, CH3, or VH region, or more than one such region, of the antibody.Alternatively, the epitope is taken from the CH2 domain of the Fc regionand transferred to the C_(L) region or V_(L) region, or both, of theantibody fragment. See also WO 97/34631 and WO 96/32478 which describeFc variants and their interaction with the salvage receptor.

Mutation of residues within Fc receptor binding sites may result inaltered effector function, such as altered ADCC or CDC activity, oraltered half-life. Potential mutations include insertion, deletion orsubstitution of one or more residues, including substitution withalanine, a conservative substitution, a non-conservative substitution,or replacement with a corresponding amino acid residue at the sameposition from a different IgG subclass (e.g., replacing an IgG1 residuewith a corresponding IgG2 residue at that position). For example, it hasbeen reported that mutating the serine at amino acid position 241 inIgG4 to proline (found at that position in IgG1 and IgG2) led to theproduction of a homogeneous antibody, as well as extending serumhalf-life and improving tissue distribution compared to the originalchimeric IgG4. (Angal et al., Mol. Immunol. 30:105-8, 1993).

In some embodiments is a pharmaceutical composition comprising anantibody comprising an ultralong CDR3; and a pharmaceutically acceptablecarrier. The antibody may comprise a therapeutic polypeptide, orderivative or variant thereof. The therapeutic polypeptide may be anon-antibody sequence. The therapeutic polypeptide, or derivative orvariant thereof can be within the ultralong CDR3. In some instances, thetherapeutic polypeptide is Moka1, Vm24, GLP-1, Exendin-4, human EPO,human FGF21, human GMCSF, human interferon-beta, human GCSF, bovine GCSFor derivative or variant thereof. Alternatively, the antibody is animmunoconjugate as described herein. The antibody can comprise one ormore immunoglobulin domains. In some embodiments, the immunoglobulindomain is an immunoglobulin A, an immunoglobulin D, an immunoglobulin E,an immunoglobulin G, or an immunoglobulin M. The immunoglobulin domaincan be an immunoglobulin heavy chain region or fragment thereof. In someinstances, the immunoglobulin domain is from a mammalian antibody.Alternatively, the immunoglobulin domain is from a chimeric antibody. Insome instances, the immunoglobulin domain is from an engineered antibodyor recombinant antibody. In other instances, the immunoglobulin domainis from a humanized, human engineered or fully human antibody. Themammalian antibody may be a bovine antibody. The mammalin antibody maybe a human antibody. In other instances, the mammalian antibody is amurine antibody. The ultralong CDR3 can comprise at least a portion of aknob domain in the CDR3. The therapeutic polypeptide can be attached tothe knob domain. Alternatively, or additionally, the ultralong CDR3comprises at least a portion of a stalk domain in the CDR3. Thetherapeutic polypeptide may be attached to the stalk domain. In someinstances, the antibody further comprises a linker. The linker can bewithin the ultralong CDR3. The linker can attach the therapeuticpolypeptide to the immunoglobulin domain or fragment thereof. In otherinstances, the linker attaches the therapeutic polypeptide to the knobdomain or stalk domain. In certain embodiments is a method of preventingor treating a disease in a subject in need thereof comprisingadministering this pharmaceutical composition to the subject. In someembodiments, the pharmaceutical composition comprising an immunoglobulinconstruct comprising a heavy chain polypeptide comprising a sequencebased on or derived from a sequence selected from any one of SEQ ID NOS:24-44 and the polypeptide sequence encoded by the DNA any one of SEQ IDNOS: 2-22; and a light chain polypeptide comprising a sequence selectedfrom SEQ ID NO: 23 and a polypeptide sequence encoded by the DNA of SEQID NO: 1; and a pharmaceutically acceptable carrier. In certainembodiments is a method of preventing or treating a disease in a mammalin need thereof comprising administering this phartmaceuticalcomposition to the mammal. In some embodiments, the disease is aninfectious disease such as mastitis. In certain embodiments, the mammalin need is a dairy animal selected from a list comprising cow, camel,donkey, goat, horse, reindeer, sheep, water buffalo, moose and yak. Insome embodiments, the mammal in need is bovine.

In some embodiments, the pharmaceutical compositions disclosed hereinmay be useful for providing prognostic or providing diagnosticinformation.

Kits/Articles of Manufacture

As an additional aspect, the present disclosure includes kits whichcomprise one or more compounds or compositions packaged in a mannerwhich facilitates their use to practice methods of the presentdisclosure. In one embodiment, such a kit includes a compound orcomposition described herein (e.g., a composition comprising an antibodycomprising an ultralong CDR3 alone or in combination with a secondagent), packaged in a container with a label affixed to the container ora package insert that describes use of the compound or composition inpracticing the method. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody comprising an ultralong CDR3 as disclosed herein;and (b) a second container with a composition contained therein, whereinthe composition comprises a further therapeutic agent. The article ofmanufacture in this embodiment disclosed herein may further comprise apackage insert indicating that the first and second compositions can beused to treat a particular condition. Alternatively, or additionally,the article of manufacture may further comprise a second (or third)container comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes. Preferably, thecompound or composition is packaged in a unit dosage form. The kit mayfurther include a device suitable for administering the compositionaccording to a specific route of administration or for practicing ascreening assay. Preferably, the kit contains a label that describes useof the antibody comprising an ultralong CDR3 composition.

In certain embodiments, the composition comprising the antibody isformulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for intravenous administration to mammals, such ashumans, bovines, felines, canines, and murines. Typically, compositionsfor intravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The amount of the composition described herein which will be effectivein the treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a Therapeuticprotein can be determined by standard clinical techniques. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses areextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

The following are examples of the methods and compositions of thedisclosure. It is understood that various other embodiments may bepracticed, given the general description provided above.

Methods of Treatment

Further disclosed herein are methods of preventing or treating a diseaseor condition in a subject in need thereof comprising administering acomposition comprising one or more antibodies comprising an ultralongCDR3 as disclosed herein to said subject. The composition can furthercomprise a pharmaceutically acceptable carrier. The subject may be amammal. The mammal may be a human. Alternatively, the mammal is abovine. The antibody may comprise a therapeutic polypeptide, orderivative or variant thereof. The therapeutic polypeptide can beencoded by a non-antibody sequence. The therapeutic polypeptide, orderivative or variant thereof can be attached to the immunoglobulindomain. The therapeutic polypeptide, or derivative or variant thereofmay be within the ultralong CDR3. Alternatively, the therapeuticpolypeptide, or derivative or variant thereof is conjugated to theultralong CDR3. In some instances, the therapeutic polypeptide is Moka1,Vm24, human GLP-1, Exendin-4, human EPO, human FGF21, human GMCSF, humaninterferon-beta, or derivative or variant thereof. The antibody may bean immunoconjugate as described herein. The antibody can comprise one ormore immunoglobulin domains. The immunoglobulin domain may be animmunoglobulin A, an immunoglobulin D, an immunoglobulin E, animmunoglobulin G, or an immunoglobulin M. The immunoglobulin domain canbe an immunoglobulin heavy chain region or fragment thereof. In someinstances, the immunoglobulin domain is from a mammalian antibody.Alternatively, the immunoglobulin domain is from a chimeric antibody.The immunoglobulin domain may be from an engineered antibody orrecombinant antibody. The immunoglobulin domain may be from a humanized,human engineered or fully human antibody. The mammalian antibody can bea bovine antibody. The mammalin antibody may be a human antibody. Inother instances, the mammalian antibody is a murine antibody. Theultralong CDR3 may be 35 amino acids in length or more. The ultralongCDR3 may comprise at least 3 cysteine residues or more. The ultralongCDR3 may comprise one or more cysteine motifs. The one or more cysteinemotifs may be based on or derived from SEQ ID NOS: 45-156. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS: 45-99.The one or more cysteine motifs may be based on or derived from SEQ IDNOS: 100-135. The one or more cysteine motifs may be based on or derivedfrom SEQ ID NOS: 136-156. The ultralong CDR3 can comprise at least aportion of a knob domain. The knob domain may comprise a conserved motifwithin the knob domain of an ultralong CDR3. For example, the knobdomain may comprise a cysteine motif disclosed herein. The therapeuticpolypeptide can be attached to the knob domain. Alternatively, oradditionally, the ultralong CDR3 comprises at least a portion of a stalkdomain. The stalk domain may comprise a conserved motif within the stalkdomain of an ultralong CDR3. The conserved motif within the stalk domainof the ultralong CDR3 may comprise a polypeptide sequence based on orderived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The conservedmotif with the stalk domain of the ultralong CDR3 may comprise apolypeptide sequence based on or derived from SEQ ID NOS: 157-224. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 157-161. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 223-234. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 235-239. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 296-299. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from a first sequenceselected from the derived from SEQ ID NOS: 300-303. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence basegroup comprising SEQ ID NOS: 157-234 and a second sequenceselected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS:333-336. The antibodies disclosed herein may comprise 2 or more, 3 ormore, 4 or more, 5 or more sequences based on or derived from SEQ IDNOS: 157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. Forexample, the stalk domain may comprise a T(S/T)VHQ motif. Thetherapeutic polypeptide can be attached to the stalk domain. In someinstances, the antibody, antibody fragment or immunoglobulin constructfurther comprises a linker. The linker can attach the therapeuticpolypeptide to the immunoglobulin domain or fragment thereof. In otherinstances, the linker attaches the therapeutic polypeptide to the knobdomain or stalk domain. In some instances, the disease or condition isan autoimmune disease, heteroimmune disease or condition, inflammatorydisease, pathogenic infection, thromboembolic disorder, respiratorydisease or condition, metabolic disease, central nervous system (CNS)disorder, bone disease or cancer. In other instances, the disease orcondition is a blood disorder. In some instances, the disease orcondition is obesity, diabetes, osteoporosis, anemia, or pain.

In some embodiments is a method of preventing or treating a disease orcondition in a subject in need thereof comprising administering to thesubject a composition comprising: an immunoglobulin construct comprisinga heavy chain polypeptide comprising a sequence that is substantiallysimilar to a sequence selected from SEQ ID NOS: 24-44; and a light chainpolypeptide comprising the sequence that is substantially similar to asequence of SEQ ID NO: 23. The heavy chain polypeptide sequence mayshare 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more amino acidsequence identity to a heavy chain sequence provided by any one of SEQID NOS: 24-44. The light chain polypeptide sequence may share 50%, 60%,70%, 80%, 85%, 90%, 95%, 97%, 99%, or more amino acid sequence identityto a light chain sequence provided by SEQ ID NO: 23. In some instances,the disease or condition is an autoimmune disease, heteroimmune diseaseor condition, inflammatory disease, pathogenic infection, thromboembolicdisorder, respiratory disease or condition, metabolic disease, centralnervous system (CNS) disorder, bone disease or cancer. In otherinstances, the disease or condition is a blood disorder. In someinstances, the disease or condition is obesity, diabetes, osteoporosis,anemia, or pain.

In an embodiment is provided a method of preventing or treating adisease or condition in a subject in need thereof comprisingadministering to the subject a composition comprising: an immunoglobulinconstruct comprising a heavy chain polypeptide comprising a polypeptidesequence encoded by a DNA sequence that is substantially similar to asequence selected from SEQ ID NOS: 2-22; and a light chain polypeptidecomprising a polypeptide sequence encoded by a DNA sequence that issubstantially similar to a sequence of SEQ ID NO: 1. The heavy chainnucleotide sequence may share 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%,99%, or more homology to a heavy chain sequence provided by any one ofSEQ ID NOS: 2-22. The light chain nucleotide sequence may share 50%,60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or more homology to a lightchain sequence provided by SEQ ID NO: 1. In some instances, the diseaseor condition is an autoimmune disease, heteroimmune disease orcondition, inflammatory disease, pathogenic infection, thromboembolicdisorder, respiratory disease or condition, metabolic disease, centralnervous system (CNS) disorder, bone disease or cancer. In otherinstances, the disease or condition is a blood disorder. In someinstances, the disease or condition is obesity, diabetes, osteoporosis,anemia, or pain.

Disclosed herein in some embodiments is a method of preventing ortreating an autoimmune disease in a subject in need thereof comprisingadministering a composition comprising one or more antibodies comprisingan ultralong CDR3 as disclosed herein to said subject. The compositioncan further comprise a pharmaceutically acceptable carrier. The subjectmay be a mammal. The mammal may be a human. Alternatively, the mammal isa bovine. The antibody may comprise a therapeutic polypeptide, orderivative or variant thereof. The therapeutic polypeptide can beencoded by a non-antibody sequence. The therapeutic polypeptide, orderivative or variant thereof can be attached to the immunoglobulindomain. The therapeutic polypeptide, or derivative or variant thereofmay be within the ultralong CDR3. Alternatively, the therapeuticpolypeptide, or derivative or variant thereof is conjugated to theultralong CDR3. In some instances, the therapeutic polypeptide is Moka1,VM-24 or beta-interferon or derivative or variant thereof. The antibodymay be an immunoconjugate as described herein. The antibody can compriseone or more immunoglobulin domains. The immunoglobulin domain may be animmunoglobulin A, an immunoglobulin D, an immunoglobulin E, animmunoglobulin G, or an immunoglobulin M. The immunoglobulin domain canbe an immunoglobulin heavy chain region or fragment thereof. In someinstances, the immunoglobulin domain is from a mammalian antibody.Alternatively, the immunoglobulin domain is from a chimeric antibody.The immunoglobulin domain may be from an engineered antibody orrecombinant antibody. The immunoglobulin domain may be from a humanized,human engineered or fully human antibody. The mammalian antibody can bea bovine antibody. The mammalin antibody may be a human antibody. Inother instances, the mammalian antibody is a murine antibody. Theultralong CDR3 may be 35 amino acids in length or more. The ultralongCDR3 may comprise at least 3 cysteine residues or more. The ultralongCDR3 may comprise one or more cysteine motifs. The one or more cysteinemotifs may be based on or derived from SEQ ID NOS: 45-156. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS: 45-99.The one or more cysteine motifs may be based on or derived from SEQ IDNOS: 100-135. The one or more cysteine motifs may be based on or derivedfrom SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least aportion of a knob domain. The knob domain may comprise a conserved motifwithin the knob domain of an ultralong CDR3. For example, the knobdomain may comprise a cysteine motif disclosed herein. The Moka1, VM-24,beta-interferon, or a derivative or variant thereof can be attached tothe knob domain. Alternatively, or additionally, the ultralong CDR3comprises at least a portion of a stalk domain. The stalk domain maycomprise a conserved motif within the stalk domain of an ultralong CDR3.The conserved motif within the stalk domain of the ultralong CDR3 maycomprise a polypeptide sequence based on or derived from SEQ ID NOS:157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 157-224. The conserved motif with thestalk domain of the ultralong CDR3 may comprise a polypeptide sequencebased on or derived from SEQ ID NOS: 157-161. The conserved motif withthe stalk domain of the ultralong CDR3 may comprise a polypeptidesequence based on or derived from SEQ ID NOS: 223-234. The conservedmotif with the stalk domain of the ultralong CDR3 may comprise apolypeptide sequence based on or derived from SEQ ID NOS: 235-239. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 296-299. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from a first sequenceselected from the derived from SEQ ID NOS: 300-303. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence basegroup comprising SEQ ID NOS: 157-234 and a second sequenceselected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS:333-336. The antibodies disclosed herein may comprise 2 or more, 3 ormore, 4 or more, 5 or more sequences based on or derived from SEQ IDNOS: 157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. Forexample, the stalk domain may comprise a T(S/T)VHQ motif. The Moka1,VM-24, beta-interferon, or a derivative or variant thereof can beattached to the stalk domain. In some instances, the antibody, antibodyfragment or immunoglobulin construct further comprises a linker. Thelinker can attach Moka1, VM-24, beta-interferon, or a derivative orvariant thereof to the immunoglobulin domain or fragment thereof. Inother instances, the linker attaches Moka1, VM-24, beta-interferon, or aderivative or variant thereof to the knob domain or stalk domain. Insome instances, the autoimmune disease is a T-cell mediated autoimmunedisease. T-cell mediated autoimmune diseases include, but are notlimited to, multiple sclerosis, type-1 diabetes, and psoriasis. In otherinstances, the autoimmune disease lupus, Sjogren's syndrome,scleroderma, rheumatoid arthritis, dermatomyositis, Hasmimoto'sthyroiditis, Addison's disease, celiac disease, Crohn's disease,pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolyticanemia, idiopathic thrombocytopenic purpura, myasthenia gravis, Ord'sthyroiditis, Graves' disease, Guillain-Barre syndrome, acutedisseminated encephalomyelitis, opsoclonus-myoclonus syndrome,ankylosing spondylitisis, antiphospholipid antibody syndrome, aplasticanemia, autoimmune hepatitis, Goodpasture's syndrome, Reiter's syndrome,Takayasu's arteritis, temporal arteritis, Wegener's granulomatosis,alopecia universalis, Behcet's disease, chronic fatigue, dysautonomia,endometriosis, interstitial cystitis, neuromyotonia, scleroderma, andvulvodynia. Lupus can include, but is not limited to, acute cutaneouslupus erythematosus, subacute cutaneous lupus erythematosus, chroniccutaneous lupus erythematosus, discoid lupus erythematosus, childhooddiscoid lupus erythematosus, generalized discoid lupus erythematosus,localized discoid lupus erythematosus, chilblain lupus erythematosus(hutchinson), lupus erythematosus-lichen planus overlap syndrome, lupuserythematosus panniculitis (lupus erythematosus profundus), tumid lupuserythematosus, verrucous lupus erythematosus (hypertrophic lupuserythematosus), complement deficiency syndromes, drug-induced lupuserythematosus, neonatal lupus erythematosus, and systemic lupuserythematosus.

Further disclosed herein is a method of preventing or treating a diseaseor condition which would benefit from the modulation of a potassiumvoltage-gated channel in a subject in need thereof comprisingadministering a composition comprising one or more antibodies comprisingan ultralong CDR3 as disclosed herein to said subject. The compositioncan further comprise a pharmaceutically acceptable carrier. In someinstances, the potassium voltage-gated channel is a KCNA3 or K_(v)1.3channel. The subject may be a mammal. The mammal may be a human.Alternatively, the mammal is a bovine. The antibody may comprise atherapeutic polypeptide, or derivative or variant thereof. Thetherapeutic polypeptide can be encoded by a non-antibody sequence. Thetherapeutic polypeptide, or derivative or variant thereof can beattached to the immunoglobulin domain. The therapeutic polypeptide, orderivative or variant thereof may be within the ultralong CDR3.Alternatively, the therapeutic polypeptide, or derivative or variantthereof is conjugated to the ultralong CDR3. In some instances, thetherapeutic polypeptide is Moka1, VM-24, or derivative or variantthereof. The antibody may be an immunoconjugate as described herein. Theantibody can comprise one or more immunoglobulin domains. Theimmunoglobulin domain may be an immunoglobulin A, an immunoglobulin D,an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. Theimmunoglobulin domain can be an immunoglobulin heavy chain region orfragment thereof. In some instances, the immunoglobulin domain is from amammalian antibody. Alternatively, the immunoglobulin domain is from achimeric antibody. The immunoglobulin domain may be from an engineeredantibody or recombinant antibody. The immunoglobulin domain may be froma humanized, human engineered or fully human antibody. The mammalianantibody can be a bovine antibody. The mammalin antibody may be a humanantibody. In other instances, the mammalian antibody is a murineantibody. The ultralong CDR3 may be 35 amino acids in length or more.The ultralong CDR3 may comprise at least 3 cysteine residues or more.The ultralong CDR3 may comprise one or more cysteine motifs. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS: 45-156.The one or more cysteine motifs may be based on or derived from SEQ IDNOS: 45-99. The one or more cysteine motifs may be based on or derivedfrom SEQ ID NOS: 100-135. The one or more cysteine motifs may be basedon or derived from SEQ ID NOS: 136-156. The ultralong CDR3 comprises atleast a portion of a knob domain. The knob domain may comprise aconserved motif within the knob domain of an ultralong CDR3. Forexample, the knob domain may comprise a cysteine motif disclosed herein.The Moka1, VM-24, or a derivative or variant thereof can be attached tothe knob domain. Alternatively, or additionally, the ultralong CDR3comprises at least a portion of a stalk domain. The stalk domain maycomprise a conserved motif within the stalk domain of an ultralong CDR3.The conserved motif within the stalk domain of the ultralong CDR3 maycomprise a polypeptide sequence based on or derived from SEQ ID NOS:157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 157-224. The conserved motif with thestalk domain of the ultralong CDR3 may comprise a polypeptide sequencebased on or derived from SEQ ID NOS: 157-161. The conserved motif withthe stalk domain of the ultralong CDR3 may comprise a polypeptidesequence based on or derived from SEQ ID NOS: 223-234. The conservedmotif with the stalk domain of the ultralong CDR3 may comprise apolypeptide sequence based on or derived from SEQ ID NOS: 235-239. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 296-299. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from a first sequenceselected from the derived from SEQ ID NOS: 300-303. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence basegroup comprising SEQ ID NOS: 157-234 and a second sequenceselected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS:333-336. The antibodies disclosed herein may comprise 2 or more, 3 ormore, 4 or more, 5 or more sequences based on or derived from SEQ IDNOS: 157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. Forexample, the stalk domain may comprise a T(S/T)VHQ motif. The Moka1,VM-24, or a derivative or variant thereof can be attached to the stalkdomain. In some instances, the antibody, antibody fragment orimmunoglobulin construct further comprises a linker. The linker canattach Moka1, VM-24, or a derivative or variant thereof to theimmunoglobulin domain or fragment thereof. In other instances, thelinker attaches Moka1, VM-24, beta-interferon, or a derivative orvariant thereof to the knob domain or stalk domain. In some instances,the disease or condition is an autoimmune disease. The autoimmunedisease can be a T-cell mediated autoimmune disease. In some instances,modulating a potassium voltage-gated channel comprises inhibiting orblocking a potassium voltage-gated channel. In some instances, thedisease or condition is episodic ataxia, seizure, or neuromyotonia.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering a composition comprising one or more antibodies comprisingan ultralong CDR3 as disclosed herein to said subject. The compositioncan further comprise a pharmaceutically acceptable carrier. The subjectmay be a mammal. The mammal may be a human. Alternatively, the mammal isa bovine. The antibody may comprise a therapeutic polypeptide, orderivative or variant thereof. The therapeutic polypeptide can beencoded by a non-antibody sequence. The therapeutic polypeptide, orderivative or variant thereof can be attached to the immunoglobulindomain. The therapeutic polypeptide, or derivative or variant thereofmay be within the ultralong CDR3. Alternatively, the therapeuticpolypeptide, or derivative or variant thereof is conjugated to theultralong CDR3. In some instances, the therapeutic polypeptide is GLP-1,Exendin-4, FGF21, or derivative or variant thereof. The GLP-1 may be ahuman GLP-1. In some instances, the FGF21 is a human FGF21. The antibodymay be an immunoconjugate as described herein. The antibody can compriseone or more immunoglobulin domains. The immunoglobulin domain may be animmunoglobulin A, an immunoglobulin D, an immunoglobulin E, animmunoglobulin G, or an immunoglobulin M. The immunoglobulin domain canbe an immunoglobulin heavy chain region or fragment thereof. In someinstances, the immunoglobulin domain is from a mammalian antibody.Alternatively, the immunoglobulin domain is from a chimeric antibody.The immunoglobulin domain may be from an engineered antibody orrecombinant antibody. The immunoglobulin domain may be from a humanized,human engineered or fully human antibody. The mammalian antibody can bea bovine antibody. The mammalin antibody may be a human antibody. Inother instances, the mammalian antibody is a murine antibody. Theultralong CDR3 may be 35 amino acids in length or more. The ultralongCDR3 may comprise at least 3 cysteine residues or more. The ultralongCDR3 may comprise one or more cysteine motifs. The one or more cysteinemotifs may be based on or derived from SEQ ID NOS: 45-156. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS: 45-99.The one or more cysteine motifs may be based on or derived from SEQ IDNOS: 100-135. The one or more cysteine motifs may be based on or derivedfrom SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least aportion of a knob domain. The knob domain may comprise a conserved motifwithin the knob domain of an ultralong CDR3. For example, the knobdomain may comprise a cysteine motif disclosed herein. The GLP-1,Exendin-4, FGF21, or a derivative or variant thereof can be attached tothe knob domain. Alternatively, or additionally, the ultralong CDR3comprises at least a portion of a stalk domain. The stalk domain maycomprise a conserved motif within the stalk domain of an ultralong CDR3.The conserved motif within the stalk domain of the ultralong CDR3 maycomprise a polypeptide sequence based on or derived from SEQ ID NOS:157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 157-224. The conserved motif with thestalk domain of the ultralong CDR3 may comprise a polypeptide sequencebased on or derived from SEQ ID NOS: 157-161. The conserved motif withthe stalk domain of the ultralong CDR3 may comprise a polypeptidesequence based on or derived from SEQ ID NOS: 223-234. The conservedmotif with the stalk domain of the ultralong CDR3 may comprise apolypeptide sequence based on or derived from SEQ ID NOS: 235-239. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 296-299. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from a first sequenceselected from the derived from SEQ ID NOS: 300-303. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence basegroup comprising SEQ ID NOS: 157-234 and a second sequenceselected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS:333-336. The antibodies disclosed herein may comprise 2 or more, 3 ormore, 4 or more, 5 or more sequences based on or derived from SEQ IDNOS: 157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. Forexample, the stalk domain may comprise a T(S/T)VHQ motif The GLP-1,Exendin-4, FGF21, or a derivative or variant thereof can be attached tothe stalk domain. In some instances, the antibody, antibody fragment orimmunoglobulin construct further comprises a linker. The linker canattach GLP-1, Exendin-4, FGF21, or a derivative or variant thereof tothe immunoglobulin domain or fragment thereof. In other instances, thelinker attaches GLP-1, Exendin-4, FGF21, or a derivative or variantthereof to the knob domain or stalk domain. Metabolic diseases and/orconditions can include disorders of carbohydrate metabolism, amino acidmetabolism, organic acid metabolism (organic acidurias), fatty acidoxidation and mitochondrial metabolism, porphyrin metabolism, purine orpyrimidine metabolism, steroid metabolism, mitochondrial function,peroxisomal function, urea cycle disorder, urea cycle defects orlysosomal storage disorders. In some instances, the metabolic disease orcondition is diabetes. In other instances, the metabolic disese orcondition is glycogen storage disease, phenylketonuria, maple syrupurine disease, glutaric acidemia type 1, Carbamoyl phosphate synthetaseI deficiency, alcaptonuria, Medium-chain acyl-coenzyme A dehydrogenasedeficiency (MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome,lipoid congenital adrenal hyperplasia, congenital adrenal hyperplasia,Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or NiemannPick disease.

Provided herein is a method of preventing or treating a central nervoussystem (CNS) disorder in a subject in need thereof comprisingadministering a composition comprising one or more antibodies comprisingan ultralong CDR3 as disclosed herein to said subject. The compositioncan further comprise a pharmaceutically acceptable carrier. The subjectmay be a mammal. The mammal may be a human. Alternatively, the mammal isa bovine. The antibody may comprise a therapeutic polypeptide, orderivative or variant thereof. The therapeutic polypeptide can beencoded by a non-antibody sequence. The therapeutic polypeptide, orderivative or variant thereof can be attached to the immunoglobulindomain. The therapeutic polypeptide, or derivative or variant thereofmay be within the ultralong CDR3. Alternatively, the therapeuticpolypeptide, or derivative or variant thereof is conjugated to theultralong CDR3. In some instances, the therapeutic polypeptide is GLP-1,Exendin-4, or derivative or variant thereof. The GLP-1 may be a humanGLP-1. The antibody may be an immunoconjugate as described herein. Theantibody can comprise one or more immunoglobulin domains. Theimmunoglobulin domain may be an immunoglobulin A, an immunoglobulin D,an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. Theimmunoglobulin domain can be an immunoglobulin heavy chain region orfragment thereof. In some instances, the immunoglobulin domain is from amammalian antibody. Alternatively, the immunoglobulin domain is from achimeric antibody. The immunoglobulin domain may be from an engineeredantibody or recombinant antibody. The immunoglobulin domain may be froma humanized, human engineered or fully human antibody. The mammalianantibody can be a bovine antibody. The mammalin antibody may be a humanantibody. In other instances, the mammalian antibody is a murineantibody. The ultralong CDR3 may be 35 amino acids in length or more.The ultralong CDR3 may comprise at least 3 cysteine residues or more.The ultralong CDR3 may comprise one or more cysteine motifs. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS: 45-156.The one or more cysteine motifs may be based on or derived from SEQ IDNOS: 45-99. The one or more cysteine motifs may be based on or derivedfrom SEQ ID NOS: 100-135. The one or more cysteine motifs may be basedon or derived from SEQ ID NOS: 136-156. The ultralong CDR3 comprises atleast a portion of a knob domain. The knob domain may comprise aconserved motif within the knob domain of an ultralong CDR3. Forexample, the knob domain may comprise a cysteine motif disclosed herein.The GLP-1, Exendin-4, or a derivative or variant thereof can be attachedto the knob domain. Alternatively, or additionally, the ultralong CDR3comprises at least a portion of a stalk domain. The stalk domain maycomprise a conserved motif within the stalk domain of an ultralong CDR3.The conserved motif within the stalk domain of the ultralong CDR3 maycomprise a polypeptide sequence based on or derived from SEQ ID NOS:157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 157-224. The conserved motif with thestalk domain of the ultralong CDR3 may comprise a polypeptide sequencebased on or derived from SEQ ID NOS: 157-161. The conserved motif withthe stalk domain of the ultralong CDR3 may comprise a polypeptidesequence based on or derived from SEQ ID NOS: 223-234. The conservedmotif with the stalk domain of the ultralong CDR3 may comprise apolypeptide sequence based on or derived from SEQ ID NOS: 235-239. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 296-299. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from a first sequenceselected from the derived from SEQ ID NOS: 300-303. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence basegroup comprising SEQ ID NOS: 157-234 and a second sequenceselected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS:333-336. The antibodies disclosed herein may comprise 2 or more, 3 ormore, 4 or more, 5 or more sequences based on or derived from SEQ IDNOS: 157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. Forexample, the stalk domain may comprise a T(S/T)VHQ motif. The GLP-1,Exendin-4, or a derivative or variant thereof can be attached to thestalk domain. In some instances, the antibody, antibody fragment orimmunoglobulin construct further comprises a linker. The linker canattach GLP-1, Exendin-4, or a derivative or variant thereof to theimmunoglobulin domain or fragment thereof. In other instances, thelinker attaches GLP-1, Exendin-4, or a derivative or variant thereof tothe knob domain or stalk domain. In some instances, the CNS disorder isAlzheimer's disease (AD). Additional CNS disorders include, but are notlimited to, encephalitis, meningitis, tropical spastic paraparesis,arachnoid cysts, Huntington's disease, locked-in syndrome, Parkinson'sdisease, Tourette's, and multiple sclerosis.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering acomposition comprising one or more antibodies comprising an ultralongCDR3 as disclosed herein to said subject. The composition can furthercomprise a pharmaceutically acceptable carrier. The subject may be amammal. The mammal may be a human. Alternatively, the mammal is abovine. The antibody may comprise a therapeutic polypeptide, orderivative or variant thereof. The therapeutic polypeptide can beencoded by a non-antibody sequence. The therapeutic polypeptide, orderivative or variant thereof can be attached to the immunoglobulindomain. The therapeutic polypeptide, or derivative or variant thereofmay be within the ultralong CDR3. Alternatively, the therapeuticpolypeptide, or derivative or variant thereof is conjugated to theultralong CDR3. In some instances, the therapeutic polypeptide is GLP-1,Exendin-4, or derivative or variant thereof. The GLP-1 may be a humanGLP-1. The antibody may be an immunoconjugate as described herein. Theantibody can comprise one or more immunoglobulin domains. Theimmunoglobulin domain may be an immunoglobulin A, an immunoglobulin D,an immunoglobulin E, an immunoglobulin G, or an immunoglobulin M. Theimmunoglobulin domain can be an immunoglobulin heavy chain region orfragment thereof. In some instances, the immunoglobulin domain is from amammalian antibody. Alternatively, the immunoglobulin domain is from achimeric antibody. The immunoglobulin domain may be from an engineeredantibody or recombinant antibody. The immunoglobulin domain may be froma humanized, human engineered or fully human antibody. The mammalianantibody can be a bovine antibody. The mammalin antibody may be a humanantibody. In other instances, the mammalian antibody is a murineantibody. The ultralong CDR3 may be 35 amino acids in length or more.The ultralong CDR3 may comprise at least 3 cysteine residues or more.The ultralong CDR3 may comprise one or more cysteine motifs. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS: 45-156.The one or more cysteine motifs may be based on or derived from SEQ IDNOS: 45-99. The one or more cysteine motifs may be based on or derivedfrom SEQ ID NOS: 100-135. The one or more cysteine motifs may be basedon or derived from SEQ ID NOS: 136-156. The ultralong CDR3 comprises atleast a portion of a knob domain. The knob domain may comprise aconserved motif within the knob domain of an ultralong CDR3. Forexample, the knob domain may comprise a cysteine motif disclosed herein.The GLP-1, Exendin-4, or a derivative or variant thereof can be attachedto the knob domain. Alternatively, or additionally, the ultralong CDR3comprises at least a portion of a stalk domain. The stalk domain maycomprise a conserved motif within the stalk domain of an ultralong CDR3.The conserved motif within the stalk domain of the ultralong CDR3 maycomprise a polypeptide sequence based on or derived from SEQ ID NOS:157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 157-224. The conserved motif with thestalk domain of the ultralong CDR3 may comprise a polypeptide sequencebased on or derived from SEQ ID NOS: 157-161. The conserved motif withthe stalk domain of the ultralong CDR3 may comprise a polypeptidesequence based on or derived from SEQ ID NOS: 223-234. The conservedmotif with the stalk domain of the ultralong CDR3 may comprise apolypeptide sequence based on or derived from SEQ ID NOS: 235-239. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 296-299. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from a first sequenceselected from the derived from SEQ ID NOS: 300-303. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence basegroup comprising SEQ ID NOS: 157-234 and a second sequenceselected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS:333-336. The antibodies disclosed herein may comprise 2 or more, 3 ormore, 4 or more, 5 or more sequences based on or derived from SEQ IDNOS: 157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. Forexample, the stalk domain may comprise a T(S/T)VHQ motif. The GLP-1,Exendin-4, or a derivative or variant thereof can be attached to thestalk domain. In some instances, the antibody, antibody fragment orimmunoglobulin construct further comprises a linker. The linker canattach GLP-1, Exendin-4, or a derivative or variant thereof to theimmunoglobulin domain or fragment thereof. In other instances, thelinker attaches GLP-1, Exendin-4, or a derivative or variant thereof tothe knob domain or stalk domain. The disease or condition can be ametabolic disease or disorder. In some instances, the disease orcondition is diabetes. In other instances, the disease or condition isobesity. Additional diseases and/or conditions which benefit from aGLP-1R and/or GCGR agonist include, but are not limited to,dyslipidemia, cardiovascular and fatty liver diseases.

Provided herein is a method of preventing or treating a blood disorderin a subject in need thereof comprising administering a compositioncomprising one or more antibodies comprising an ultralong CDR3 asdisclosed herein to said subject. The composition can further comprise apharmaceutically acceptable carrier. The subject may be a mammal. Themammal may be a human. Alternatively, the mammal is a bovine. Theantibody may comprise a therapeutic polypeptide, or derivative orvariant thereof. The therapeutic polypeptide can be encoded by anon-antibody sequence. The therapeutic polypeptide, or derivative orvariant thereof can be attached to the immunoglobulin domain. Thetherapeutic polypeptide, or derivative or variant thereof may be withinthe ultralong CDR3. Alternatively, the therapeutic polypeptide, orderivative or variant thereof is conjugated to the ultralong CDR3. Insome instances, the therapeutic polypeptide is erythropoietin, GMCSF, orderivative or variant thereof. The erythropoietin may be a humanerythropoietin. The GMCSF may be a human GMCSF. The antibody may be animmunoconjugate as described herein. The antibody can comprise one ormore immunoglobulin domains. The immunoglobulin domain may be animmunoglobulin A, an immunoglobulin D, an immunoglobulin E, animmunoglobulin G, or an immunoglobulin M. The immunoglobulin domain canbe an immunoglobulin heavy chain region or fragment thereof. In someinstances, the immunoglobulin domain is from a mammalian antibody.Alternatively, the immunoglobulin domain is from a chimeric antibody.The immunoglobulin domain may be from an engineered antibody orrecombinant antibody. The immunoglobulin domain may be from a humanized,human engineered or fully human antibody. The mammalian antibody can bea bovine antibody. The mammalin antibody may be a human antibody. Inother instances, the mammalian antibody is a murine antibody. Theultralong CDR3 may be 35 amino acids in length or more. The ultralongCDR3 may comprise at least 3 cysteine residues or more. The ultralongCDR3 may comprise one or more cysteine motifs. The one or more cysteinemotifs may be based on or derived from SEQ ID NOS: 45-156. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS: 45-99.The one or more cysteine motifs may be based on or derived from SEQ IDNOS: 100-135. The one or more cysteine motifs may be based on or derivedfrom SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least aportion of a knob domain. The knob domain may comprise a conserved motifwithin the knob domain of an ultralong CDR3. For example, the knobdomain may comprise a cysteine motif disclosed herein. Theerythropoietin, GMCSF, or a derivative or variant thereof can beattached to the knob domain. Alternatively, or additionally, theultralong CDR3 comprises at least a portion of a stalk domain. The stalkdomain may comprise a conserved motif within the stalk domain of anultralong CDR3. The conserved motif within the stalk domain of theultralong CDR3 may comprise a polypeptide sequence based on or derivedfrom SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence based on or derived from SEQ ID NOS: 157-224. The conservedmotif with the stalk domain of the ultralong CDR3 may comprise apolypeptide sequence based on or derived from SEQ ID NOS: 157-161. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 223-234. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 235-239. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 296-299. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from a first sequenceselected from the derived from SEQ ID NOS: 300-303. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence basegroup comprising SEQ ID NOS: 157-234 and a second sequenceselected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS:333-336. The antibodies disclosed herein may comprise 2 or more, 3 ormore, 4 or more, 5 or more sequences based on or derived from SEQ IDNOS: 157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. Forexample, the stalk domain may comprise a T(S/T)VHQ motif. Theerythropoietin, GMCSF, or a derivative or variant thereof can beattached to the stalk domain. In some instances, the antibody, antibodyfragment or immunoglobulin construct further comprises a linker. Thelinker can attach erythropoietin, GMCSF, or a derivative or variantthereof to the immunoglobulin domain or fragment thereof. In otherinstances, the linker attaches erythropoietin, GMCSF, or a derivative orvariant thereof to the knob domain or stalk domain. In some instances,the blood disorder is anemia. Examples of anemia include, but are notlimited to, herditary xerocytosis, congenital dyserythropoietic anemia,Rh null disease, infectious mononucleosis related anemia, drugs-relatedanemia, aplastic anemia, microcytic anemia, macrocytic anemia,normocytic anemia, hemolytic anemia, poikilocytic anemia, spherocyticanemia, drepanocytic anemia, normochromic anemia, hyperchromic anemia,hypochromic anemia, macrocytic-normochromic anemia,microcytic-hypochromic anemia, normocytic-normochromic anemia,iron-deficiency anemia, pernicious anemia, folate-deficiency anemia,thalassemia, sideroblastic anemia, posthemorrhagic anemia, sickle cellanemia, chronic anemia, achrestic anemia, autoimmune haemolytic anemia,Cooley's anemia, drug-induced immune haemolytic anemia, erythroblasticanemia, hypoplastic anemia, Diamond-Blackfan anemia, Pearson's anemia,transient anemia, Fanconi's anemia, Lederer's anemia, myelpathic anemia,nutritional anemia, spur-cell anemia, Von Jaksh's anemia, sideroblaticanemia, sideropenic anemia, alpha thalassemia, beta thalassemia,hemoglobin h disease, acute acquired hemolytic anemia, warm autoimmunehemolytic anemia, cold autoimmune hemolytic anemia, primary coldautoimmune hemolytic anemia, secondary cold autoimmune hemolytic anemia,secondary autoimmune hemolytic anemia, primary autoimmune hemolyticanemia, x-linked sideroblastic anemia, pyridoxine-responsive anemia,nutritional sideroblastic anemia, pyridoxine deficiency-inducedsideroblastic anemia, copper deficiency-induced sideroblastic anemia,cycloserine-induced sideroblastic anemia, chloramphenicol-inducedsideroblastic anemia, ethanol-induced sideroblastic anemia,isoniazid-induced sideroblastic anemia, drug-induced sideroblasticanemia, toxin-induced sideroblastic anemia, microcytic hyperchromicanemia, macrocytic hyperchromic anemia, megalocytic-normochromic anemia,drug-induced immune hemolytic anemia, non-hereditary spherocytic anemia,inherited spherocytic anemia, and congenital spherocytic anemia. Inother instances, the blood disorder is malaria. Alternatively, the blooddisorder is lymphoma, leukemia, multiple myeloma, or myelodysplasticsyndrome. In some instances, the blood disorder is neutropenia,Shwachmann-Daimond syndrome, Kostmann syndrome, chronic granulomatousdisease, leukocyte adhesion deficiency, meyloperoxidase deficiency, orChediak Higashi syndrome.

Provided herein is a method of preventing or treating a disease ordisorder which benefitis from stimulating or increasing white blood cellproduction in a subject in need thereof comprising administering acomposition comprising one or more antibodies comprising an ultralongCDR3 as disclosed herein to said subject. The composition can furthercomprise a pharmaceutically acceptable carrier. The subject may be amammal. The mammal may be a human. Alternatively, the mammal is abovine. The antibody may comprise a therapeutic polypeptide, orderivative or variant thereof. The therapeutic polypeptide can beencoded by a non-antibody sequence. The therapeutic polypeptide, orderivative or variant thereof can be attached to the immunoglobulindomain. The therapeutic polypeptide, or derivative or variant thereofmay be within the ultralong CDR3. Alternatively, the therapeuticpolypeptide, or derivative or variant thereof is conjugated to theultralong CDR3. In some instances, the therapeutic polypeptide is GMCSF,or derivative or variant thereof. The GMCSF may be a human GMCSF. Theantibody may be an immunoconjugate as described herein. The antibody cancomprise one or more immunoglobulin domains. The immunoglobulin domainmay be an immunoglobulin A, an immunoglobulin D, an immunoglobulin E, animmunoglobulin G, or an immunoglobulin M. The immunoglobulin domain canbe an immunoglobulin heavy chain region or fragment thereof. In someinstances, the immunoglobulin domain is from a mammalian antibody.Alternatively, the immunoglobulin domain is from a chimeric antibody.The immunoglobulin domain may be from an engineered antibody orrecombinant antibody. The immunoglobulin domain may be from a humanized,human engineered or fully human antibody. The mammalian antibody can bea bovine antibody. The mammalin antibody may be a human antibody. Inother instances, the mammalian antibody is a murine antibody. Theultralong CDR3 may be 35 amino acids in length or more. The ultralongCDR3 may comprise at least 3 cysteine residues or more. The ultralongCDR3 may comprise one or more cysteine motifs. The one or more cysteinemotifs may be based on or derived from SEQ ID NOS: 45-156. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS: 45-99.The one or more cysteine motifs may be based on or derived from SEQ IDNOS: 100-135. The one or more cysteine motifs may be based on or derivedfrom SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least aportion of a knob domain. The knob domain may comprise a conserved motifwithin the knob domain of an ultralong CDR3. For example, the knobdomain may comprise a cysteine motif disclosed herein. The GMCSF, or aderivative or variant thereof can be attached to the knob domain.Alternatively, or additionally, the ultralong CDR3 comprises at least aportion of a stalk domain. The stalk domain may comprise a conservedmotif within the stalk domain of an ultralong CDR3. The conserved motifwithin the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence based on or derived from SEQ ID NOS: 157-307 and SEQ ID NOS:333-336. The conserved motif with the stalk domain of the ultralong CDR3may comprise a polypeptide sequence based on or derived from SEQ ID NOS:157-224. The conserved motif with the stalk domain of the ultralong CDR3may comprise a polypeptide sequence based on or derived from SEQ ID NOS:157-161. The conserved motif with the stalk domain of the ultralong CDR3may comprise a polypeptide sequence based on or derived from SEQ ID NOS:223-234. The conserved motif with the stalk domain of the ultralong CDR3may comprise a polypeptide sequence based on or derived from SEQ ID NOS:235-239. The conserved motif with the stalk domain of the ultralong CDR3may comprise a polypeptide sequence based on or derived from SEQ ID NOS:296-299. The conserved motif with the stalk domain of the ultralong CDR3may comprise a polypeptide sequence based on or derived from SEQ ID NOS:300-303. The conserved motif with the stalk domain of the ultralong CDR3may comprise a polypeptide sequence based on or derived from a firstsequence selected from the derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence basegroup comprising SEQ ID NOS: 157-234 and asecond sequence selected from the group comprising SEQ ID NOS: 235-307and SEQ ID NOS: 333-336. The antibodies disclosed herein may comprise 2or more, 3 or more, 4 or more, 5 or more sequences based on or derivedfrom SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence based on or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS:333-336. For example, the stalk domain may comprise a T(S/T)VHQ motif.The GMCSF, or a derivative or variant thereof can be attached to thestalk domain. In some instances, the antibody, antibody fragment orimmunoglobulin construct further comprises a linker. The linker canattach GMCSF, or a derivative or variant thereof to the immunoglobulindomain or fragment thereof. In other instances, the linker attachesGMCSF, or a derivative or variant thereof to the knob domain or stalkdomain. In some instances, the disese or disorder is neutropenia,Shwachmann-Daimond syndrome, Kostmann syndrome, chronic granulomatousdisease, leukocyte adhesion deficiency, meyloperoxidase deficiency, orChediak Higashi syndrome.

Provided herein is a method of preventing or treating a disease ordisorder which benefitis from stimulating or increasing red blood cellproduction in a subject in need thereof comprising administering acomposition comprising one or more antibodies comprising an ultralongCDR3 as disclosed herein to said subject. The composition can furthercomprise a pharmaceutically acceptable carrier. The subject may be amammal. The mammal may be a human. Alternatively, the mammal is abovine. The antibody may comprise a therapeutic polypeptide, orderivative or variant thereof. The therapeutic polypeptide can beencoded by a non-antibody sequence. The therapeutic polypeptide, orderivative or variant thereof can be attached to the immunoglobulindomain. The therapeutic polypeptide, or derivative or variant thereofmay be within the ultralong CDR3. Alternatively, the therapeuticpolypeptide, or derivative or variant thereof is conjugated to theultralong CDR3. In some instances, the therapeutic polypeptide iserythropoietin, or derivative or variant thereof. The erythropoietin maybe a human erythropoietin. The antibody may be an immunoconjugate asdescribed herein. The antibody can comprise one or more immunoglobulindomains. The immunoglobulin domain may be an immunoglobulin A, animmunoglobulin D, an immunoglobulin E, an immunoglobulin G, or animmunoglobulin M. The immunoglobulin domain can be an immunoglobulinheavy chain region or fragment thereof. In some instances, theimmunoglobulin domain is from a mammalian antibody. Alternatively, theimmunoglobulin domain is from a chimeric antibody. The immunoglobulindomain may be from an engineered antibody or recombinant antibody. Theimmunoglobulin domain may be from a humanized, human engineered or fullyhuman antibody. The mammalian antibody can be a bovine antibody. Themammalin antibody may be a human antibody. In other instances, themammalian antibody is a murine antibody. The ultralong CDR3 may be 35amino acids in length or more. The ultralong CDR3 may comprise at least3 cysteine residues or more. The ultralong CDR3 may comprise one or morecysteine motifs. The one or more cysteine motifs may be based on orderived from SEQ ID NOS: 45-156. The one or more cysteine motifs may bebased on or derived from SEQ ID NOS: 45-99. The one or more cysteinemotifs may be based on or derived from SEQ ID NOS: 100-135. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS:136-156. The ultralong CDR3 comprises at least a portion of a knobdomain. The knob domain may comprise a conserved motif within the knobdomain of an ultralong CDR3. For example, the knob domain may comprise acysteine motif disclosed herein. The erythropoietin, or a derivative orvariant thereof can be attached to the knob domain. Alternatively, oradditionally, the ultralong CDR3 comprises at least a portion of a stalkdomain. The stalk domain may comprise a conserved motif within the stalkdomain of an ultralong CDR3. The conserved motif within the stalk domainof the ultralong CDR3 may comprise a polypeptide sequence based on orderived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The conservedmotif with the stalk domain of the ultralong CDR3 may comprise apolypeptide sequence based on or derived from SEQ ID NOS: 157-224. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 157-161. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 223-234. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 235-239. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 296-299. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from a first sequenceselected from the derived from SEQ ID NOS: 300-303. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence basegroup comprising SEQ ID NOS: 157-234 and a second sequenceselected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS:333-336. The antibodies disclosed herein may comprise 2 or more, 3 ormore, 4 or more, 5 or more sequences based on or derived from SEQ IDNOS: 157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. Forexample, the stalk domain may comprise a T(S/T)VHQ motif. Theerythropoietin, or a derivative or variant thereof can be attached tothe stalk domain. In some instances, the antibody, antibody fragment orimmunoglobulin construct further comprises a linker. The linker canattach erythropoietin, or a derivative or variant thereof to theimmunoglobulin domain or fragment thereof. In other instances, thelinker attaches erythropoietin, or a derivative or variant thereof tothe knob domain or stalk domain. In some instances, the disease ordisorder is anemia.

Provided herein is a method of preventing or treating obesity in asubject in need thereof comprising administering a compositioncomprising one or more antibodies comprising an ultralong CDR3 asdisclosed herein to said subject. The composition can further comprise apharmaceutically acceptable carrier. The subject may be a mammal. Themammal may be a human. Alternatively, the mammal is a bovine. Theantibody may comprise a therapeutic polypeptide, or derivative orvariant thereof. The therapeutic polypeptide can be encoded by anon-antibody sequence. The therapeutic polypeptide, or derivative orvariant thereof can be attached to the immunoglobulin domain. Thetherapeutic polypeptide, or derivative or variant thereof may be withinthe ultralong CDR3. Alternatively, the therapeutic polypeptide, orderivative or variant thereof is conjugated to the ultralong CDR3. Insome instances, the therapeutic polypeptide is GLP-1, Exendin-4, FGF21,or derivative or variant thereof. The GLP-1 may be a human GLP-1. Insome instances, the FGF21 is a human FGF21. The antibody may be animmunoconjugate as described herein. The antibody can comprise one ormore immunoglobulin domains. The immunoglobulin domain may be animmunoglobulin A, an immunoglobulin D, an immunoglobulin E, animmunoglobulin G, or an immunoglobulin M. The immunoglobulin domain canbe an immunoglobulin heavy chain region or fragment thereof. In someinstances, the immunoglobulin domain is from a mammalian antibody.Alternatively, the immunoglobulin domain is from a chimeric antibody.The immunoglobulin domain may be from an engineered antibody orrecombinant antibody. The immunoglobulin domain may be from a humanized,human engineered or fully human antibody. The mammalian antibody can bea bovine antibody. The mammalin antibody may be a human antibody. Inother instances, the mammalian antibody is a murine antibody. Theultralong CDR3 may be 35 amino acids in length or more. The ultralongCDR3 may comprise at least 3 cysteine residues or more. The ultralongCDR3 may comprise one or more cysteine motifs. The one or more cysteinemotifs may be based on or derived from SEQ ID NOS: 45-156. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS: 45-99.The one or more cysteine motifs may be based on or derived from SEQ IDNOS: 100-135. The one or more cysteine motifs may be based on or derivedfrom SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least aportion of a knob domain. The knob domain may comprise a conserved motifwithin the knob domain of an ultralong CDR3. For example, the knobdomain may comprise a cysteine motif disclosed herein. The GLP-1,Exendin-4, FGF21, or a derivative or variant thereof can be attached tothe knob domain. Alternatively, or additionally, the ultralong CDR3comprises at least a portion of a stalk domain. The stalk domain maycomprise a conserved motif within the stalk domain of an ultralong CDR3.The conserved motif within the stalk domain of the ultralong CDR3 maycomprise a polypeptide sequence based on or derived from SEQ ID NOS:157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 157-224. The conserved motif with thestalk domain of the ultralong CDR3 may comprise a polypeptide sequencebased on or derived from SEQ ID NOS: 157-161. The conserved motif withthe stalk domain of the ultralong CDR3 may comprise a polypeptidesequence based on or derived from SEQ ID NOS: 223-234. The conservedmotif with the stalk domain of the ultralong CDR3 may comprise apolypeptide sequence based on or derived from SEQ ID NOS: 235-239. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 296-299. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from a first sequenceselected from the derived from SEQ ID NOS: 300-303. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence basegroup comprising SEQ ID NOS: 157-234 and a second sequenceselected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS:333-336. The antibodies disclosed herein may comprise 2 or more, 3 ormore, 4 or more, 5 or more sequences based on or derived from SEQ IDNOS: 157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. Forexample, the stalk domain may comprise a T(S/T)VHQ motif. The GLP-1,Exendin-4, FGF21, or a derivative or variant thereof can be attached tothe stalk domain. In some instances, the antibody, antibody fragment orimmunoglobulin construct further comprises a linker. The linker canattach GLP-1, Exendin-4, FGF21, or a derivative or variant thereof tothe immunoglobulin domain or fragment thereof. In other instances, thelinker attaches GLP-1, Exendin-4, FGF21, or a derivative or variantthereof to the knob domain or stalk domain.

Provided herein is a method of preventing or treating a pain in asubject in need thereof comprising administering a compositioncomprising one or more antibodies, antibody fragments, or immunoglobulinconstructs described herein to said subject. In some instances, thesubject is a mammal. In certain instances, the mammal is a human.Alternatively, the mammal is a bovine. In some instances, the one ormore antibodies, antibody fragments, or immunoglobulin constructscomprise a protoxin2 or a derivative or variant thereof. Alternatively,or additionally, the one or more antibodies, antibody fragments, orimmunoglobulin constructs comprise at least a portion of a CDR3H. Theportion of the CDR3H can be a stalk domain or knob domain in the CDR3H.In some instances, the one or more antibodies, antibody fragments, orimmunoglobulin constructs further comprise a linker. The linker canattach the protoxin2 or a derivative or variant thereof to the portionof the CDR3H.

Provided herein is a method of preventing or treating a disease orcondition which benefits from modulating a sodium ion channel in asubject in need thereof comprising administering a compositioncomprising one or more antibodies, antibody fragments, or immunoglobulinconstructs described herein to said subject. In some instances, thesubject is a mammal. In certain instances, the mammal is a human.Alternatively, the mammal is a bovine. In some instances, the one ormore antibodies, antibody fragments, or immunoglobulin constructscomprise a protoxin2 or a derivative or variant thereof. Alternatively,or additionally, the one or more antibodies, antibody fragments, orimmunoglobulin constructs comprise at least a portion of a CDR3H. Theportion of the CDR3H can be a stalk domain or knob domain in the CDR3H.In some instances, the one or more antibodies, antibody fragments, orimmunoglobulin constructs further comprise a linker. The linker canattach the protoxin2 or a derivative or variant thereof to the portionof the CDR3H. In some instances, the sodium ion channel is a Na_(v)channel. In some instances, the Na_(c) channel is a Na_(v)1.7 channel.In some instances, modulating a sodium ion channel comprises inhibitingor blocking a sodium ion channel. In some instances, the disease orcondition is Dravet Syndrome, generalized epilepsy with febrile seizuresplus (GEFS+), paramyotonia congenital or erythromelalgia. In someinstances, the disease or condition is pain.

Provided herein is a method of preventing or treating a disease orcondition which benefits from modulating an acid sensing ion channel(ASIC) in a subject in need thereof comprising administering acomposition comprising one or more antibodies, antibody fragments, orimmunoglobulin constructs described herein to said subject. In someinstances, the subject is a mammal. In certain instances, the mammal isa human. Alternatively, the mammal is a bovine. In some instances, theone or more antibodies, antibody fragments, or immunoglobulin constructscomprise a protoxin2 or a derivative or variant thereof. Alternatively,or additionally, the one or more antibodies, antibody fragments, orimmunoglobulin constructs comprise at least a portion of a CDR3H. Theportion of the CDR3H can be a stalk domain or knob domain in the CDR3H.In some instances, the one or more antibodies, antibody fragments, orimmunoglobulin constructs further comprise a linker. The linker canattach the protoxin2 or a derivative or variant thereof to the portionof the CDR3H. In some instances, modulating an ASIC comprises inhibitingor blocking the ASIC. In some instances, the disease or condition is acentral nervous system disorder. In other instances, the disease orcondition is pain.

Provided herein is a method of preventing or treating a pathogenicinfection in a subject in need thereof comprising administering acomposition comprising one or more antibodies comprising an ultralongCDR3 as disclosed herein to said subject. The composition can furthercomprise a pharmaceutically acceptable carrier. The subject may be amammal. The mammal may be a human. Alternatively, the mammal is abovine. The antibody may comprise a therapeutic polypeptide, orderivative or variant thereof. The therapeutic polypeptide can beencoded by a non-antibody sequence. The therapeutic polypeptide, orderivative or variant thereof can be attached to the immunoglobulindomain. The therapeutic polypeptide, or derivative or variant thereofmay be within the ultralong CDR3. Alternatively, the therapeuticpolypeptide, or derivative or variant thereof is conjugated to theultralong CDR3. In some instances, the therapeutic polypeptide isbeta-interferon, or derivative or variant thereof. The antibody may bean immunoconjugate as described herein. The antibody can comprise one ormore immunoglobulin domains. The immunoglobulin domain may be animmunoglobulin A, an immunoglobulin D, an immunoglobulin E, animmunoglobulin G, or an immunoglobulin M. The immunoglobulin domain canbe an immunoglobulin heavy chain region or fragment thereof. In someinstances, the immunoglobulin domain is from a mammalian antibody.Alternatively, the immunoglobulin domain is from a chimeric antibody.The immunoglobulin domain may be from an engineered antibody orrecombinant antibody. The immunoglobulin domain may be from a humanized,human engineered or fully human antibody. The mammalian antibody can bea bovine antibody. The mammalin antibody may be a human antibody. Inother instances, the mammalian antibody is a murine antibody. Theultralong CDR3 may be 35 amino acids in length or more. The ultralongCDR3 may comprise at least 3 cysteine residues or more. The ultralongCDR3 may comprise one or more cysteine motifs. The one or more cysteinemotifs may be based on or derived from SEQ ID NOS: 45-156. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS: 45-99.The one or more cysteine motifs may be based on or derived from SEQ IDNOS: 100-135. The one or more cysteine motifs may be based on or derivedfrom SEQ ID NOS: 136-156. The ultralong CDR3 comprises at least aportion of a knob domain. The knob domain may comprise a conserved motifwithin the knob domain of an ultralong CDR3. For example, the knobdomain may comprise a cysteine motif disclosed herein. Thebeta-interferon, or a derivative or variant thereof can be attached tothe knob domain. Alternatively, or additionally, the ultralong CDR3comprises at least a portion of a stalk domain. The stalk domain maycomprise a conserved motif within the stalk domain of an ultralong CDR3.The conserved motif within the stalk domain of the ultralong CDR3 maycomprise a polypeptide sequence based on or derived from SEQ ID NOS:157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 157-224. The conserved motif with thestalk domain of the ultralong CDR3 may comprise a polypeptide sequencebased on or derived from SEQ ID NOS: 157-161. The conserved motif withthe stalk domain of the ultralong CDR3 may comprise a polypeptidesequence based on or derived from SEQ ID NOS: 223-234. The conservedmotif with the stalk domain of the ultralong CDR3 may comprise apolypeptide sequence based on or derived from SEQ ID NOS: 235-239. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 296-299. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from a first sequenceselected from the derived from SEQ ID NOS: 300-303. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence basegroup comprising SEQ ID NOS: 157-234 and a second sequenceselected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS:333-336. The antibodies disclosed herein may comprise 2 or more, 3 ormore, 4 or more, 5 or more sequences based on or derived from SEQ IDNOS: 157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. Forexample, the stalk domain may comprise a T(S/T)VHQ motif. Thebeta-interferon, or a derivative or variant thereof can be attached tothe stalk domain. In some instances, the antibody, antibody fragment orimmunoglobulin construct further comprises a linker. The linker canattach beta-interferon, or a derivative or variant thereof to theimmunoglobulin domain or fragment thereof. In other instances, thelinker attaches beta-interferon, or a derivative or variant thereof tothe knob domain or stalk domain. In some instances, the pathogenicinfection is a viral, bacterial, fungal, or parasitic infection. In someinstances, the viral infection is a herpes virus.

Provided herein is a method of preventing or treating a cancer in asubject in need thereof comprising administering a compositioncomprising one or more antibodies comprising an ultralong CDR3 asdisclosed herein to said subject. The composition can further comprise apharmaceutically acceptable carrier. The subject may be a mammal. Themammal may be a human. Alternatively, the mammal is a bovine. Theantibody may comprise a therapeutic polypeptide, or derivative orvariant thereof. The therapeutic polypeptide can be encoded by anon-antibody sequence. The therapeutic polypeptide, or derivative orvariant thereof can be attached to the immunoglobulin domain. Thetherapeutic polypeptide, or derivative or variant thereof may be withinthe ultralong CDR3. Alternatively, the therapeutic polypeptide, orderivative or variant thereof is conjugated to the ultralong CDR3. Insome instances, the therapeutic polypeptide is beta-interferon, orderivative or variant thereof. The antibody may be an immunoconjugate asdescribed herein. The antibody can comprise one or more immunoglobulindomains. The immunoglobulin domain may be an immunoglobulin A, animmunoglobulin D, an immunoglobulin E, an immunoglobulin G, or animmunoglobulin M. The immunoglobulin domain can be an immunoglobulinheavy chain region or fragment thereof. In some instances, theimmunoglobulin domain is from a mammalian antibody. Alternatively, theimmunoglobulin domain is from a chimeric antibody. The immunoglobulindomain may be from an engineered antibody or recombinant antibody. Theimmunoglobulin domain may be from a humanized, human engineered or fullyhuman antibody. The mammalian antibody can be a bovine antibody. Themammalin antibody may be a human antibody. In other instances, themammalian antibody is a murine antibody. The ultralong CDR3 may be 35amino acids in length or more. The ultralong CDR3 may comprise at least3 cysteine residues or more. The ultralong CDR3 may comprise one or morecysteine motifs. The one or more cysteine motifs may be based on orderived from SEQ ID NOS: 45-156. The one or more cysteine motifs may bebased on or derived from SEQ ID NOS: 45-99. The one or more cysteinemotifs may be based on or derived from SEQ ID NOS: 100-135. The one ormore cysteine motifs may be based on or derived from SEQ ID NOS:136-156. The ultralong CDR3 comprises at least a portion of a knobdomain. The knob domain may comprise a conserved motif within the knobdomain of an ultralong CDR3. For example, the knob domain may comprise acysteine motif disclosed herein. The beta-interferon, or a derivative orvariant thereof can be attached to the knob domain. Alternatively, oradditionally, the ultralong CDR3 comprises at least a portion of a stalkdomain. The stalk domain may comprise a conserved motif within the stalkdomain of an ultralong CDR3. The conserved motif within the stalk domainof the ultralong CDR3 may comprise a polypeptide sequence based on orderived from SEQ ID NOS: 157-307 and SEQ ID NOS: 333-336. The conservedmotif with the stalk domain of the ultralong CDR3 may comprise apolypeptide sequence based on or derived from SEQ ID NOS: 157-224. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 157-161. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 223-234. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 235-239. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 296-299. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from SEQ ID NOS: 300-303. Theconserved motif with the stalk domain of the ultralong CDR3 may comprisea polypeptide sequence based on or derived from a first sequenceselected from the derived from SEQ ID NOS: 300-303. The conserved motifwith the stalk domain of the ultralong CDR3 may comprise a polypeptidesequence basegroup comprising SEQ ID NOS: 157-234 and a second sequenceselected from the group comprising SEQ ID NOS: 235-307 and SEQ ID NOS:333-336. The antibodies disclosed herein may comprise 2 or more, 3 ormore, 4 or more, 5 or more sequences based on or derived from SEQ IDNOS: 157-307 and SEQ ID NOS: 333-336. The conserved motif with the stalkdomain of the ultralong CDR3 may comprise a polypeptide sequence basedon or derived from SEQ ID NOS: 304-307 AND SEQ ID NOS: 333-336. Forexample, the stalk domain may comprise a T(S/T)VHQ motif. Thebeta-interferon, or a derivative or variant thereof can be attached tothe stalk domain. In some instances, the antibody, antibody fragment orimmunoglobulin construct further comprises a linker. The linker canattach beta-interferon, or a derivative or variant thereof to theimmunoglobulin domain or fragment thereof. In other instances, thelinker attaches beta-interferon, or a derivative or variant thereof tothe knob domain or stalk domain. In some instances, the cancer is ahematological malignancy. The hematological malignancy can be a leukemiaor lymphoma. In some instances, the hematological malignancy is a B-celllymphoma, T-cell lymphoma, follicular lymphoma, marginal zone lymphoma,hairy cell leukemia, chronic myeloid leukemia, mantle cell lymphoma,nodular lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, chroniclymphocytic leukemia, or small lymphocytic leukemia.

Provided herein is a method of preventing or treating a disease in amammal in need thereof comprising administering a pharmaceuticalcomposition described herein to said mammal. In some embodiments, thedisease is an infectious disease. In certain embodiments, the infectiousdisease is mastitis. In some embodiments, the infectious disease is arespiratory disease. In certain embodiments, the respiratory disease isbovine respiratory disease of shipping fever. In certain embodiments,the mammal in need is a dairy animal selected from a list comprisingcow, camel, donkey, goat, horse, reindeer, sheep, water buffalo, mooseand yak. In some embodiments, the mammal in need is bovine.

Provided is a method of preventing or treating mastitis in a dairyanimal, comprising providing to said dairy animal an effective amount ofa composition comprising: an immunoglobulin construct comprising a heavychain polypeptide comprising a sequence selected from SEQ ID NO: 25 andSEQ ID NO: 26; and a light chain polypeptide comprising the sequence ofSEQ ID NO: 23. In an embodiment is provided a method of preventing ortreating mastitis in a dairy animal, comprising providing to said dairyanimal an effective amount of a composition comprising: animmunoglobulin construct comprising a heavy chain polypeptide comprisinga polypeptide sequence encoded by the DNA selected from SEQ ID NO: 4 andSEQ ID NO: 5; and a light chain polypeptide comprising a polypeptidesequence encoded by the DNA of SEQ ID NO: 1. In some embodiments, thedairy animal is a cow or a water buffalo. Provided are methods oftreatment, inhibition and prevention by administration to a subject ofan effective amount of an antibody or pharmaceutical compositiondescribed herein. The antibody may be substantially purified (e.g.,substantially free from substances that limit its effect or produceundesired side-effects). The subject can be an animal, including but notlimited to animals such as cows, pigs, sheep, goats, rabbits, horses,chickens, cats, dogs, mice, etc. The subject can be a mammal. In someinstances, the subject is a human. Alternatively, the subject is abovine.

Various delivery systems are known and can be used to administer anantibody formulation described herein, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, in certain embodiments, it is desirableto introduce the heteromultimer compositions described herein into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir.

In a specific embodiment, it is desirable to administer the antibody, orcompositions described herein locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion, topical application, e.g., in conjunction with a wounddressing after surgery, by injection, by means of a catheter, by meansof a suppository, or by means of an implant, said implant being of aporous, non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers. Preferably, when administering aprotein, including an antibody, of the invention, care must be taken touse materials to which the protein does not absorb.

In another embodiment, the antibody or pharmaceutical composition isdelivered in a vesicle, in particular a liposome (see Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; seegenerally ibid.)

In yet another embodiment, the heteromultimers or composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl.J. Med. 321:574 (1989)). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190(1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,e.g., the brain, thus requiring only a fraction of the systemic dose(see, e.g., Goodson, in Medical Applications of Controlled Release,supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems arediscussed in the review by Langer (Science 249:1527-1533 (1990)).

In a specific embodiment comprising a nucleic acid encoding a antibodydescribed herein, the nucleic acid can be administered in vivo topromote expression of its encoded protein, by constructing it as part ofan appropriate nucleic acid expression vector and administering it sothat it becomes intracellular, e.g., by use of a retroviral vector (seeU.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

In some embodiments are crystals based on or derived from the antibodiesdisclosed herein. The crystals may have a space group P2₁2₁2₁. In someinstances, the crystal has the unit cell dimensions of “a” between about40 to 80 angstroms, between 45 to about 75 angstroms, or between about50 to about 75 angstroms; “b” between about 40 to 140 angstroms, betweenabout 50 to about 130 angstroms, between about 55 to about 130angstroms; and “c” between 100 to about 350 angstroms, between 120 toabout 340 angstroms, or between about 125 to about 330 angstroms.Alternatively, the crystal has the unit cell dimensions of “a” greaterthan or equal to 40, 50, 60, 70, or 80 angstroms; “b” greater than orequal to 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 angstroms; and“c” greater than or equal to 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, or 340 angstroms.

The crystal may comprise a bovine antibody or portion thereof. Thecrystal may comprise a Fab fragment based on or derived from a bovineantibody. The crystal may comprise a non-antibody sequence, linker,cleave site, non-bovine sequence, or a combination thereof. The crystalmay be an isolated crystal. The antibody may be based on or derived fromthe peptide sequence of any of SEQ ID NOS: 1-44. The antibody maycomprise at least a portion of a heavy chain. The portion of the heavychain may comprise the peptide sequence of any of SEQ ID NOS: 24-44. Theantibody may comprise at least a portion of a heavy chain. The portionof the heavy chain may be encoded by a DNA sequence based on or derivedfrom the DNA sequence of any of SEQ ID NOS: 2-22. The antibody maycomprise at least a portion of a light chain. The portion of the lightchain may comprise the peptide sequence of SEQ ID NO: 23. The antibodymay comprise at least a portion of a heavy chain. The portion of theheavy chain may be encoded by a DNA sequence based on or derived fromthe DNA sequence of SEQ ID NOS: 1.

In some embodiments, is an isolated crystal comprising a bovine antibodyFab fragment comprising SEQ ID NO: 24 and SEQ ID NO: 23, wherein thecrystal has a space group P2₁2₁2₁ and unit cell dimensions of a=71.4angstroms, b=127.6 angstroms and c=127.9 angstroms.

In some embodiments, is an isolated crystal comprising a bovine antibodyFab fragment comprising SEQ ID NO: 340 and SEQ ID NO: 341, wherein thecrystal has a space group P2₁2₁2₁ and unit cell dimensions of a=54.6angstroms, b=53.7 angstroms and c=330.5 angstroms.

Example 1 Purification and Crystallization of Antibodies Comprising anUltralong CDR3

An antibody that comprises an ultralong CDR3 including, for example, anantibody generated by any of the examples described herein, may bepurified and subsequently crystallized to determine the structure of theantibody.

A. Purification:

Genes encoding the heavy and light chain Fab regions of BLV1H12 andBLV5B8 were generated by gene synthesis (GenScript, Piscataway, N.J.). ADNA fragment derived from the promoter region of pFastBacDual(Invitrogen) was fused to the gp67 and the honey bee mellitin (HBM)signal peptides by overlap PCR, yielding a fragment with head-to-headp10 and polyhedrin promoters upstream of the HBM and gp67 signalpeptides, respectively (i.e., HBM-p10-pPolyH-gp67). Bovine Fab heavy andlight chain regions were fused to the promoter-signal peptide cassetteby overlap PCR (heavy chain downstream of pPolyH-gp67 and light chaindownstream of p10-HBM), and ligated into the SfiI sites of pDCE361, aderivative of pFastBacDual. Next, a His6-tag was introduced at theC-terminus of the heavy chain to facilitate purification. The resultingbaculovirus transfer vectors were used to generate recombinant bacmidsusing the Bac-to-Bac system (Invitrogen) and virus was rescued bytransfecting purified bacmid DNA into Sf9 cells using Cellfectin II(Invitrogen). Both Fab proteins were produced by infecting suspensioncultures of Sf9 cells with recombinant baculovirus at an MOI of 5-10 andincubating at 28° C. with shaking at 110 RPM. After 72 hours, thecultures were clarified by two rounds of centrifugation at 2000 g and10,000 g at 4° C. The supernatant, containing secreted, soluble Fab werethen concentrated and buffer exchanged into 1×PBS, pH 7.4. After metalaffinity chromatography using Ni-NTA resin, Fabs were purified byprotein G affinity chromatography (GE Healthcare), cation exchangechromatography (MonoS, GE healthcare), and gel filtration (Superdex200,GE Healthcare).

B. Crystallization and Structure Determination

Gel filtration fractions containing the bovine Fabs were concentrated to˜10 mg/mL in 10 mM Tris, pH 8.0 and 50 mM NaCl. Initial crystallizationtrials were set up using the automated Rigaku Crystalmation roboticsystem at the Joint Center for Structural Genomics. Several hits wereobtained for BLV1H12 and BLV5B8, and crystals used for data collectionwere grown by the sitting drop vapor diffusion method with a reservoirsolution (100 μL) containing 0.27 M potassium citrate and 22% PEG 3350(BLV1H12) and 0.2 M di-sodium tartrate and 20% PEG 3350 (BLV5B8). Dropsconsisting of 100 nL protein+100 mL precipitant were set up at 20° C.,and crystals appeared within 3-7 days. The resulting crystals werecryoprotected using well solution supplemented with 15% ethylene glycolthen flash cooled and stored in liquid nitrogen until data collection.

Diffraction data were then collected on the GM/CA-CAT 23ID-D beamline atthe Advanced Photon Source at Argonne National Laboratory (BLV1H12) andthe 11-1 beamline at the Stanford Synchrotron Radiation Lightsource forBLV5B8. Both datasets were indexed in spacegroup P212121, integrated,scaled, and merged using HKL2000 (BLV5B8; HKL Research) or XPREP(BLV1H12; Bruker). The BLV1H12 structure was solved by molecularreplacement to 1.88 Å resolution using Phaser (McCoy et al., 2007). Fabvariable domains from 1BVK and constant domains from 2FB4 were used assearch models and two complete BLV1H12 Fabs were found in the asymmetricunit. The BLV5B8 dataset was also solved by molecular replacement (to2.20 Å), using the refined BLV1H12 coordinates as a model. Rigid bodyrefinement, simulated annealing and restrained refinement (including TLSrefinement, with one group for each Ig domain and one for each CDR H3)were carried out in Phenix (Adams et al. (2010) Acta Crystallogr D BiolCrystallogr 66:213-221). Riding hydrogens were used during refinementand are included in the final model.

Between rounds of refinement, the model was built and adjusted usingCoot. Waters were built automatically using the “ordered solvent”modeling function in Phenix (Adams et al. (2010) Acta Crystallogr D BiolCrystallogr 66:213-221). Structures were validated using the JCSG QCServer (publicly available at http://smb.slac.stanford.edu/jcsg/QC/),which includes Molprobity (Chen et al. (2010) Acta Crystallogr D BiolCrystallogr 66:12-21) Table 1

TABLE 1 Data collection and refinement statistics Data collectionBLV1H12 Fab BLV5B8 Fab Beamline APS 231D-D SSRL 11-1 Wavelength (Å)1.033 0.979 Space group P2₁2₁2₁ P2₁2₁2₁ Unit cell parameters a = 71.4, b= 127.6, c = 127.9, A = 54.6, b = 53.7, c = 330.5, (Å, °) α = β = γ = 90α = β = γ = 90 Resolution (Å) 50-1.88 (1.92-1.88) 50-2.20 (2.28-2.20)Observations 638,900 313,175 Unique Reflections 96,353 49,527 Redundancy6.2 (4.9) 6.3 (3.5) Completeness (%) 97.3 (98.2) 96.7 (75.4) <1/α₁> 14.7(2.5) 17.8 (2.3) R_(sym) ^(b) 0.09 (0.76) 0.10 (0.45) Z_(a) ^(c) 2 2Refinement statistics Resolution (Å) 50-1.88 50-2.20 Reflections (work)89,254 46,900 Reflections (test) 4,704 2,441R_(cryst)(%)^(d)/R_(free)(%)^(c) 20.8/23.9 2.07/24.8 Average B (Å²) 43.042.7 Wilson B (Å²) 32.5 32.0 Protein atoms 6,724 6,939 Carbohydrateatoms 0 0 Waters 501 474 Other 1 0 RMSD from ideal geometry Bond length(Å) 0.014 0.003 Bond angles (°) 1.12 0.79 Ramachandran statistics(%)^(r) Favored 96.9 95.2 Outliers 0.1 0.5 PDB Code ^(g) wwww Xxxx^(a)Numbers in parentheses refer to the highest resolution shell.^(b)R_(sym) = Σ_(hkl)Σ_(i) | I_(hkl, i) − <I_(hkl)>|/Σ_(hkl)Σ_(i)I_(hkl, I) and R_(pim) = Σ_(hkl) (1/(n − 1))^(1/2)Σ_(i) |I_(hkl, i) − <I_(hkl)> | /Σ_(hkl)Σ_(i)I_(hkl, I), where I_(hkl, i) isthe scaled intensity of the i^(th) measurement of relection h, k, l,<I_(hkl)> is the average intensity for that reflection, and n is theredundancy (Emsley et al. (2010) Acta Crystallogr D Biol Crystallogr 66:486-501). ^(c)Z_(a) is the number of Fabs per crystallographicasymmetric unit. ^(d)R_(cryst) = Σ_(hkl) | F_(o) − F_(c) |/Σ_(hkl) |F_(o) | × 100 ^(e)R_(free) was calculated as for R_(cryst), but on atest set comprising 5% of the data excluded from refinement.^(f)Calculated using Molprobity (Chen et al. (2010) Acta Crystallogr DBiol Crystallogr 66: 12-21). ^(g) Coordinates and structure factors willbe deposited in the PDB prior to publication and be availableimmediately on publication.

Example 2 Generation of Libraries of Polynucleotides Encoding AntibodiesComprising an Ultralong CDR3

Bovine spleen and lymph nodes were obtained from Animal Technologies(Tyler, Tex.), or from Texas A&M University. Total RNA was isolated frombovine tissues from three different cows (MID1, MID10, and MID 11) usingTRIzol reagent (Invitrogen, Carlsbad, Calif., USA) followed by on columndigestion of DNA using the RNeasy Mini Kit (Qiagen, Valencia, Calif.,USA). Next, RNA quantity and quality were assessed with Nanodrop(Thermal Scientific), Qubit RNA and Agilent 2100 Bioanalyzer (Agilent,Santa Clara, Calif., USA), following the manufacturer's protocols. TotalRNA was used as a template for cDNA synthesis catalyzed by SuperscriptII (Invitrogen).

The library of amplified antibody variable regions were then subjectedto deep sequencing. Briefly, bar-coded primers (Table 2) for each of thethree cows (MID1, MID10, and MID11) were used to amplify V_(H) frombovine spleen cDNA.

TABLE 2  Bar-coded primers for deep sequencing SEQ ID Description NO:Isotype Primers MID1 FW 308 IgG CCTATCCCCTGTGTGCCTTGGCAGTCTCAGACGAGTGCGTTTGAGCGACAAGGCTGTAGGCTG MID1 RV 309 IgGCCATCTCATCCCTGCGTGTCTCCGACTCAGAC GAGTGCGTCTTTCGGGGCTGTGGTGGAGGC MID10 FW310 IgM CCTATCCCCTGTGTGCCTTGGCAGTCTCAGTC TCTATGCGTTGAGCGACAAGGCTGTAGGCTGMID10 RV 311 IgM CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTCTATGCGAGTGAAGACTCTCGGGTGTGATT CAC MID11 FW 312 IgMCCTATCCCCTGTGTGCCTTGGCAGTCTCAGTG ATACGTCTTTGAGCGACAAGGCTGTAGGCTGMID11 RV 313 IgM CCATCTCATCCCTGCGTGTCTCCGACTCAGTGATACGTCTAGTGAAGACTCTCGGGTGTGATT CAC Primer A 314 TTGAGCGACAAGGCTGTAGGCTGPrimer B 315 CTTTCGGGGCTGTGGTGG-AGGC Primer C 316 AGATCCAAGCTGTGACCGGC

Next, the amplicons of V_(H) were purified from 2% agarose gels and deepsequenced according to Roche 454 GS FLX instructions. Multiplealignments were performed with the MUSCLE algorithm (Edgar (2004)Nucleic Acids Research 32:1792-1797). MUSCLE was executed to generatemultiple long CDR H3 nucleotide alignments with relatively high gap open(−20.0) and gap extend (−10.0) penalties due to the large amount ofheterogeneity observed in the sequences. Local alignment was executedusing the Smith-Waterman algorithm with the following settings, matchscore=2.0, mismatch penalty=−1.0, gap opening penalty=−2.0, and gapextension penalty=−0.5. CDR H3s were defined by the third residuefollowing the conserved cysteine in framework 3 to the residueimmediately preceding the conserved tryptophan in framework 4. V_(H)BULwas identified by BLAST searching the bovine genome (assemblyBtau_(—)4.6.1) with multiple ultralong V_(H) sequences identified bydeep sequencing. The deep sequencing identified a total of 11,728ultralong CDR3 sequences with having a length between 44 and 69 aminoacid residues. The results of the deep sequencing are summarized inTable 3 below.

TABLE 3 Summary of deep sequencing results from bovine spleen Cow#1Cow#1 Cow#2 Source (Bar code) (MID1) (MID10) (MID11) Ig Class IgG IgMIgM CDR H3 length range 44-66 44-68 44-69 Number of unique cysteinepatterns 655 449 847 Total number of unique long CDR H3 5633 1639 4456sequences

The results of the deep sequencing also revealed that ultralong CDR3comprise a cysteine motif (e.g., a pattern of cysteine residues) thatcomprises between 3 and 12 cysteine residues. Representative examples ofcysteine patterns are shown for the deep sequencing run for threedifferent cows (MID1, MID10, and MID11) as well as their abundance inthe run (Table 4-6; SEQ ID NOS: 45-156). The cysteines in the ultralongCDR3 regions are symbolized as “C”. The amino acids between twocysteines are symbolized as “X_(n)”. Additional exemplary cysteinemotifs are shown in the ultralong CDR3 sequences set forth in Table 23.

TABLE 4 Cysteine patterns identified in ultralong CDR3s from MID1Cysteine pattern (MID1) Abundance (%) CX₁₀CX₅CX₅CXCX₇C 10.44%CX₁₀CX₆CX₅CXCX₁₅C 8.11% CX₁₁CXCX₅C 5.22% CX₁₁CX₅CX₅CXCX₇C 2.56%CX₁₀CX₆CX₅CXCX₁₃C 1.47% CX₁₀CX₅CXCX₄CX₈C 1.19% CX₁₀CX₆CX₆CXCX₇C 1.08%CX₁₀CX₄CX₇CXCX₈C 1.05% CX₁₀CX₄CX₇CXCX₇C 0.91% CX₁₃CX₈CX₈C 0.91%CX₁₀CX₆CX₅CXCX₇C 0.59% CX₁₀CX₅CX₅C 0.57% CX₁₀CX₅CX₆CXCX₇C 0.50%CX₁₀CX₆CX₅CX₇CX₉C 0.43% CX₉CX₇CX₅CXCX₇C 0.41% CX₁₀CX₆CX₅CXCX₉C 0.36%CX₁₀CXCX₄CX₅CX₁₁C 0.32% CX₇CX₃CX₆CX₅CXCX₅CX₁₀C 0.32%CX₁₀CXCX₄CX₅CXCX₂CX₃C 0.30% CX₁₆CX₅CXC 0.23%

TABLE 5 Cysteine patterns identified in ultralong CDR3s from MID10Cysteine pattern (MID10) Abundance (%) CX₁₀CXCX₄CX₅CXCX₂CX₃C 2.87%CX₁₀CX₅CX₅C 0.73% CX₁₀CXCX₄CX₅CX₁₁C 0.67% CX₆CX₄CXCX₄CX₅C 0.61%CX₁₁CX₄CX₅CX₆CX₃C 0.55% CX₈CX₂CX₆CX₅C 0.43% CX₁₀CX₅CX₅CXCX₁₀C 0.37%CX₁₀CXCX₆CX₄CXC 0.31% CX₁₀CX₅CX₅CXCX₂C 0.31% CX₁₄CX₂CX₃CXCXC 0.31%CX₁₅CX₅CXC 0.31% CX₄CX₆CX₉CX₂CX₁₁C 0.31% CX₆CX₄CX₅CX₅CX₁₂C 0.31%CX₇CX₃CXCXCX₄CX₅CX₉C 0.31% CX₁₀CX₆CX₅C 0.24% CX₇CX₃CX₅CX₅CX₉C 0.24%CX₇CX₅CXCX₂C 0.24% CX₁₀CXCX₆C 0.18% CX₁₀CX₃CX₃CX₅CX₇CXCX₆C 0.18%CX₁₀CX₄CX₅CX₁₂CX₂C 0.18%

TABLE 6 Cysteine patterns identified in ultralong CDR3s from MID11Cysteine pattern (MID11) Abundance (%) CX₁₂CX₄CX₅CXCXCX₉CX₃C 1.19%CX₁₂CX₄CX₅CX₁₂CX₂C 0.96% CX₁₀CX₆CX₅CXCX₁₁C 0.92% CX₁₆CX₅CXCXCX₁₄C 0.70%CX₁₀CX₅CXCX₈CX₆C 0.52% CX₁₂CX₄CX₅CX₈CX₂C 0.49% CX₁₂CX₅CX₅CXCX₈C 0.47%CX₁₀CX₆CX₅CXCX₄CXCX₉C 0.45% CX₁₁CX₄CX₅CX₈CX₂C 0.45% CX₁₀CX₆CX₅CX₈CX₂C0.43% CX₁₀CX₆CX₅CXCX₈C 0.36% CX₁₀CX₆CX₅C 0.31% CX₁₀CX₆CX₅CXCX₃CX₈CX₂C0.29% CX₁₀CX₆CX₅CX₃CX₈C 0.29% CX₁₀CX₆CX₅CXCX₂CX₆CX₅C 0.25%CX₇CX₆CX₃CX₃CX₉C 0.25% CX₉CX₈CX₅CX₆CX₅C 0.22% CX₁₀CX₂CX₂CX₇CXCX₁₁CX₅C0.20% CX₁₀CX₆CX₅CXCX₁₃C 0.20% CX₁₀CX₆CX₅CXCX₂CX₈CX₄C 0.20%

Bovine V_(H) regions were amplified from cDNA prepared in example 9using primers 5′-TTGAGCGACAAGGCTGTAGGCTG-3′ (SEQ ID NO: 314) and5′-CTTTCGGGGCTGTGGTGG-AGGC-3′ (SEQ ID NO: 315) producing a library ofantibody variable region cDNA biased for ultralong CDRs. Next, themixture of V_(H) regions was assembled by overlap PCR with bovine C_(H)1and human IgG Fc. Briefly, EcoRI and NheI sites were incorporated forligation into pFUSE expression vector, to afford a full-length heavychain library ready for expression in mammalian cells. The ligationproduct was transformed into E. coli and 500 single E. colitransformants were picked. Each transformant was then grown overnight ina separate vessel and DNA from each colony was extracted using Qiagenminprep kits (Qiagen, Inc.) and sequenced by BATJ, Inc. (San Diego,Calif.) using the oligo 5′-AGATCCAAGCTGTGACCGGC-3′ (SEQ ID NO: 316).Sequences were analyzed using VectorNTI (Invitrogen, Inc. Carlsbad,Calif.). Duplicative sequences, sequences with no insert, and sequencesencoding a CDR shorter than 35 residues were excluded. 132 clonescontaining unique long CDR heavy chain sequences were selected. Eachheavy chain in the 132 member library was then co-transfected inparallel with pFUSE expression vector encoding the invariant bovinelight chain (SEQ ID NO: N) into 293T cells, to generate a smallspatially addressed library (Mao et al. (2010) Nat Biotech28:1195-1202). 130,000 293T cells per well were plated in 24 well platesand grown overnight in 500 ul DMEM media (Invitrogen) with 10% FBS(Invitrogen), and Penicillin/streptomycin/glutamine (Invitrogen) at 37°C. and 5% CO₂. 0.5 μg of Hc-encoding pFuse vector and 0.5 μg ofLc-encoding pFuse vector were added to 25 μl of optimem (Invitrogen). 1μl of Lipofectamine 2000 or 293Fectin transfection reagent (Invitrogen)was added to 25 μl of optimem, and incubated 5 minutes. Next, theDNA-optimem mix and transfection reagent-optimem mix were combined andincubated 15 minutes, added to 293T cells, and allowed to incubate oncells 4-6 hours. Then media was then aspirated from wells and replacedwith fresh media, and cells were allowed to grow and secrete IgG intothe media for 4 days. Cell-culture supernatants containing IgG wereharvested in 96 well format for further testing. The chimeric antibodieswere quantified by sandwich ELISA detecting human F_(c) and screened forbinding to BVDV by ELISA.

Antibodies were then secreted into culture media and harvested in a 96well format to generate a small spatially addressed library for furthertesting including, screening for binding to BVDV by ELISA. For example,an ELISA was conducted to screen the antibody library for binding toBVDV. Briefly, killed BVDV (0.2 μg) in 100 μL DPBS was coated on 96-wellMaxiSorp ELISA plates (Nunc) for 1 hour at 37° C. Next, the plates wereblocked with 200 μL 3% BSA solution in DPBST, Dulbecco's phosphatebuffered saline, 0.25% Tween 20) for 1 hour at 37° C. Samples were thenincubated with 3% BSA in DPBST for 1 hour at 37° C. Subsequently, wellswere washed 5 times with 200 μL DPBST. Next, Goat Anti-Human IgG(Fc)—HRP conjugated antibody (KPL Inc.) was added at a 1:1,000 dilutionin blocking solution and incubated for 1 hour at 37° C. Wells were thenwashed 10 times with 200 μL DPBST. A 100 μL working solution ofQuantaBlu (Pierce) was added to each well and incubated for 5 minutes atroom temperature before plates were read in a SpectraMax M5 plate readerat ex325/em420 nm. Several candidate binders were identified (FIG. 1B,left). Clone H12 has a 63-residue CDR3 with 6 cysteine residues and wasable to strongly bind BVDV in a dose dependent fashion (FIG. 1B, right;and 1C).

Additionally, binding of the chimeric recombinant antibodies to BVDVantigens was evaluated by immunocytometric analysis of transfected humanembryonic kidney (HEK) 293A cells (Invitrogen), as previously described(see, e.g., Njongmeta et al. (2012) Vaccine 30:1624-1635). Briefly, HEK293A monolayers grown in 6-well tissue culture plates were transfectedwith 2 μg/well of plasmid (pcDNA3.3, Invitrogen) encoding BVDV antigens(N^(pro), E2, or non-structural proteins NS2-3) using Lipofectamine 2000reagent (Invitrogen), and incubated for 48 hr at 37° C. with 5% CO₂. Themonolayers were fixed with ice-cold 100% methanol for 10 minutes, rinsedwith PBS, and after blocking for 1 hr with PBS containing 5% fetalbovine serum (blocking buffer), the monolayers were incubated at roomtemperature for 1 hr with 10 μg/ml of a mouse anti-FLAG M2-alkalinephosphatase (AP)-conjugate (Sigma) in blocking buffer or 10 μg/ml of thechimeric recombinant antibodies (H12 or B8). Monolayers transfected withempty vector were similarly reacted to serve as negative controls and,following washes in blocking buffer, the monolayers probed with thechimeric recombinant antibodies were incubated with a 1/200 dilution ofAP-conjugated goat anti-Human IgG (Fc specific) mAb (Sigma) in blockingbuffer for 1 hr. Following washes in blocking buffer, the AP activity inall the wells was detected using Fast Red AS-MX substrate (Sigma).Stained cells were visualized and photographed using an IS70 invertedoptical microscope (Olympus, Japan) equipped with a camera. H12 stronglybinds HEK293A cells transfected with the NS2-3 non-structural proteinsof BVDV but weakly bound to untransfected cells while B8 had weakbinding to both HEK293A cells transfected with the NS2-3 non-structuralproteins of BVDV and untransfected cells (FIG. 1D).

Example 3 Generation and Testing of Libraries of Antibodies Comprisingan Ultralong CDR3

A library of polynucleotides coding for antibodies that comprise anultralong CDR3 is generated by immunization of cattle with whole killedbovine viral diarrhea virus (BVDV) (FIG. 1A). Briefly, a four month-oldHolstein steer was immunized by intradermal inoculation of a mixture ofheat killed BVDV-1 and BVDV-2 (100 μg of each). The inactivated virusmixture was suspended in 500 μl PBS and emulsified in 500 μl Freund'sComplete Adjuvant by repeated passage through a double barrel needle.Next, the immunogen was inoculated intradermally (200 μl/injection) atthe neck region using a 26×1¹/2 G needle. The steer was then boostedthree times at monthly intervals with the same amount of antigen butformulated in Freund's Incomplete Adjuvant. Sero-conversion was testedby ELISA using plates coated with the inactivated virus and byimmunocytometric analysis of MDBK cells infected with either BVDV-1 orBVDV-2. The steer was then bled from the jugular vein and blood wascollected in heparin. Lymphocytes were purified through LymphocyteSeparation Media (Mediatech) centrifugation and stored in RNAlater. AcDNA library was then made from the plurality of lymphocyte RNA asdescribed in Example 2.

Example 4 Constructing Vectors of BLV1H12-bGCSF Fusion Proteins forExpression in Mammalian Cells

A gene encoding bovine G-CSF (bGCSF) was synthesized by Genscript (NJ,USA) and amplified by polymerase chain reaction (PCR). To optimize thefolding and stability of the immunoglobulin constructs, flexible linkersof (GGGGS)n (n=0, 1) were added on both ends of the bGCSF fragment.Subsequently, PCR fragments of bGCSF with varied lengths of linkers weregrafted into the complementarity determining region 3 of the heavy chain(CDR3H) of BLV1H12 antibody by exploiting overlap extension PCR, toreplace the ‘knob’ domain as shown in the crystal structure of BLV1H12.The expression vectors of BLV1H12-bGCSF fusion proteins were generatedby in-frame ligation of the amplified BLV1H12-bGCSF fusion genes to thepFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly, the geneencoding the light chain of BLV1H12 antibody was cloned into the pFusevector without hIgG1 Fc fragment. The obtained expression vectors wereconfirmed by sequencing. FIGS. 8F, and 8G provide depictions of thebovine G-CSF inserted into or replacing at least a portion of the knobdomain of a heavy chain region of bovine BLV1H12 antibody. FIG. 14 showsvectors for expression of the BLV1H12-bGCSF fusion protein. DNAsequences encoding the heavy and light chain immunoglobulin constructsAb-bGCSF L0 (n=0) and Ab-bGCSF L1 (n=1) are shown in Table 19. Aminoacid sequences encoding the heavy and light chain immunoglobulinconstructs Ab-bGCSF L0 and Ab-bGCSF L1 are shown in Table 21.

Example 5 Expression and Purification of BLV1H12-bGCSF Fusion Antibodies

BLV1H12-bGCSF fusion antibodies were expressed through transienttransfections of free style HEK 293 cells with vectors encodingBLV1H12-bGCSF fusion heavy chain and BLVH1H12 light chain. ExpressedBLV1H12-bGCSF fusion antibodies were secreted into the culture mediumand harvested at 48 hours and 96 hours after transfection. TheBLV1H12-bGCSF fusion antibodies were purified by Protein A/Gchromatography (Thermo Fisher Scientific, IL), and analyzed by SDS-PAGEgel. FIG. 15A shows SDS-PAGE gel of purified Ab-bGCSF L0 and Ab-bGCSF L1fusion antibodies from HEK 293 cells.

Example 6 In Vitro Study of Proliferative Activities of theBLV1H12-bGCSF Fusion Antibodies on Mouse NFS-60 Cells

Mouse NFS-60 cells were obtained from American Type Culture Collection(ATCC), VA, and cultured in RPMI-1640 medium supplemented with 10% fetalbovine serum (FBS), 0.05 mM 2-mercapoethanol and 62 ng/ml humanmacrophage colony stimulating factor (M-CSF). For proliferation assay,mouse NFS-60 cells were washed three times with RPMI-1640 medium andresuspended in RPMI-1640 medium with 10% FBS and 0.05 mM2-mercapoethanol at a density of 1.5×10⁵ cells/ml. In 96-well plates,100 μl of cell suspension was added into each well, followed by theaddition of varied concentrations of bGCSF, BLV1H12 antibody, and theimmunoglobulin constructs described herein, Equal volume of PBS bufferwas added into the control wells. The plates were incubated at 37° C. ina 5% CO₂ incubator for 72 hours. Cells were then treated with AlamarBlue(Invitrogen) ( 1/10 volume of cell suspension) for 4 hours at 37° C.Fluorescence at 595 nm for each well was read to indicate the cellviability. As seen in FIGS. 9A-9E and Tables 7-8, the immunoglobulinconstructs Ab-bGCSF L0 and Ab-bGCSF L1 display similar activity as thebovine G-CSF and human G-CSF.

TABLE 7 bGCSF hGCSF Ab (ng/mL) F.I. (ng/mL) F.I. (ng/mL) F.I. 0.091451213.67567 0.09145 1266.554 0.45725 1039.57933 0.27435 1360.925 0.274351478.287 1.37174 1009.55533 0.82305 1761.00533 0.82305 1752.216 4.11523983.27867 2.46914 2100.51733 2.46914 2224.028 12.34568 971.84967 7.407412405.09667 7.40741 2587.222 37.03704 960.54933 22.22222 2646.2406722.22222 2751.249 111.11111 991.83 66.66667 2812.098 66.66667 2815.37333.33333 964.798 200 3087.144 200 2948.509

TABLE 8 Ab-bGCSF Ab-bGCSF L1 (ng/mL) F.I. (ng/mL) F.I. 0.098761327.94667 0.09808 1412.387 0.29627 1435.92467 0.29424 1654.776 0.888811734.765 0.88272 2082.718 2.66642 2188.50333 2.64816 2550.674 7.999272680.08167 7.94449 2997.807 23.9978 2899.28333 23.83346 3277.812 71.99342920.56767 71.50039 3308.383 215.9802 3416.20433 214.50117 3868.075

Example 7 In Vitro Study of Proliferative Activities of BLV1H12-bGCSFFusion Antibodies on Human Granulocyte Progenitors

Human mPB CD34⁺ cells were purchased from AllCells. Cells wereresuspended in HSC expansion medium (StemSpan SFEM, StemCellTechnologies) supplemented with 1× antibiotics and the followingrecombinant human cytokines thrombopoietin, IL6, Flt3 ligand, and stemcell factor (100 ng/mL, R & D Systems), then plated in 96-well plates(1000 cells per well), with varied concentrations of BLV1H12-bGCSFfusion antibodies. Cells were cultured for 7 days at 37° C. in a 5% CO₂incubator, then analyzed by flow cytometry to measure cell number andexpression of CD45ra and CD41. Cells were stained in staining medium(HBSS supplemented with 2% FBS and 2 mM EDTA) at 4° C. for 1 h withPECy7 anti-CD45ra and eFluor 450 anti-CD41 (eBiosciences), then washedwith staining medium and analyzed. Multicolor analysis for cellphenotyping was performed on a LSR II flow cytometer (Becton Dickinson).FIGS. 10A-10E and Tables 9-10 show the human granulocyte progenitor cellproliferative activities of the Ab-GCSF fusion antibodies.

TABLE 9 Number of Number of Number of bGCSF CD45RA− hGCSF CD45RA− AbCD45RA− (ng/mL) CD41− (ng/mL) CD41− (ng/mL) CD41− 0.55886 7354 0.050814175 0.28959 3666 1.67657 9776 0.15242 3651 0.86877 3839 5.02972 127000.45725 3671 2.60631 3852 15.08916 13200 1.37174 4299 23.45679 351945.26749 13700 4.11523 5900 70.37037 3541 135.80247 13300 12.34568 7784211.11111 3606 407.40741 13700 37.03704 10500 5700 4100 1222.22222 14100111.11111 12200 3666.66667 13100 333.33333 14100 11000 13800 1000 13900

TABLE 10 Number of Number of Ab-bGCSF CD45RA− Ab-bGCSF L1 CD45RA−(ng/mL) CD41− (ng/mL) CD41− 0.14225 2601 0.30483 4070 0.42676 22530.91449 3601 1.28029 4155 2.74348 4303 3.84088 4399 8.23045 640411.52263 7262 24.69136 9122 34.5679 9902 74.07407 11200 103.7037 11500222.22222 11000 311.11111 11500 666.66667 11400 933.33333 11900 200011400 2800 12100 6000 13200

Example 8 Pharmacokinetics of BLV1H12-bGCSF Fusion Antibodies in Mice

For PK study in mice, 70 μg of the immunoglobulin constructs describedherein were injected into 3 BALB/c mice per group. Blood samples weredrawn from time 0 to 14 days with extended intervals and analyzed byELISA using anti-human IgG Fc antibody with horseradish peroxidase (HRP)labeled (KPL) and anti-6×His antibody with HRP labeled (Clontech). Datawere normalized by taking maximal concentration at the first time point(30 min). As seen in FIGS. 11A-11B and Table 11, the half-life forbovine G-CSF was significantly increased when provided in the form of animmunoglobulin construct described herein.

TABLE 11 bGCSF Ab Ab-GCSF L1 Ab-GCSF L0 hour Percentage day Percentageday Percentage day Percentage 0.5 100.00% 0.021 100.00% 0.021 100.00%0.021 100.00% 1 79.52% 0.042 106.09% 0.042 102.12% 0.042 91.12% 3 56.81%0.083 95.88% 0.083 104.03% 0.083 91.24% 6 14.94% 0.125 86.54% 0.125101.71% 0.125 91.56% 24 4.08% 0.250 74.26% 0.250 93.90% 0.250 61.22% 480.00% 1.000 52.43% 1.000 59.26% 1.000 44.22% 72 0.00% 2.000 39.46% 2.00056.66% 2.000 36.36% 3.000 36.97% 3.000 39.09% 3.000 27.38% 8.000 45.44%8.000 22.21% 8.000 11.53% 10.000 40.98% 10.000 21.15% 10.000 12.44%14.000 51.96% 14.000 21.41% 14.000 14.35%

Example 9 Neutrophils Counts in Mice

On the 10^(th) day after injection of BLV1H12-bGCSF fusion antibodiesinto mice for PK study, blood samples were drawn from the mice andstained using the Diff Quick Staining Kit (Thermo Fisher Scientific,IL). Neutrophils and white blood cells were counted under microscope andthe percentages of neutrophils were analyzed. FIGS. 12A-12B and Table 12show proliferative activities of BLV1H12-bGCSF fusion antibodies on miceneutrophils that are blood stained and counted at the 10th daypost-injection.

TABLE 12 Percentage of Neutrophil N.C 16.07 Ab 18.81 Ab-GCSF L1 25.32Ab-GCSF L0 25.05 bGCSF 16.48

Example 10 Construction of Vectors of BLV1H12-bGCSF Fusion Proteins forExpression in Pichia pastoris

Gene fragments encoding BLV1H12-bGCSF chain heavy chain and BLV1H12light chain were amplified from the pFuse expression vectors andsubsequently ligated into the same pPICZα vector (Invitrogen). Theexpressions of the heavy and light chains were under the control of AOX1promoter.

Example 11 Expression and Purification of BLV1H12-bGCSF FusionAntibodies in Pichia

The Pichia GS190 cells were transformed with the pPICZα vectors encodingimmunoglobulin construct heavy and light chains by electroporation.Positive transformants were selected based on zeocin resistance andconfirmed by PCR. Pichia cells with integrated BLV1H12-bGCSF fusiongenes were grown in BMGY medium till OD₆₀₀=2˜6. The cells were thentransferred into BMMY medium in ⅕ of its original volume for inductionof the proteins expression. For every 24 hours, a final concentration of0.5% methanol was added into the medium to maintain the induction.Medium containing the secreted immunoglobulin constructs were harvestedafter 96-hour induction. The BLV1H12-bGCSF fusion antibodies werepurified by Protein A/G chromatography (Thermo Fisher Scientific, IL)and analyzed by SDS-PAGE gel. FIGS. 13A-13C show expression andpurification of BLV1H12-bGCSF fusion antibodies in Pichia pastoris.

Example 12 Constructing Vectors of BLV1H12-Moka1 Fusion Proteins forExpression in Mammalian Cells

A gene encoding Moka1 was synthesized by Genscript or IDT, and amplifiedby polymerase chain reaction (PCR). To optimize the folding andstability of fusion proteins, flexible linkers of (GGGGS)n (n=0, 1) wereadded on both ends of the Moka1 fragment. Subsequently, PCR fragments ofMoka1 with and without the linker were grafted into the complementaritydetermining region 3 of the heavy chain (CDR3H) of BLV1H12 antibody byexploiting overlap extension PCR, to replace the ‘knob’ domain as shownin the crystal structure of BLV1H12. The expression vectors ofBLV1H12-Moka1 fusion proteins were generated by in-frame ligation of theamplified BLV1H12-Moka1 fusion genes to the pFuse-hIgG1-Fc backbonevector (InvivoGen, CA). Similarly, the gene encoding the light chain ofBLV1H12 antibody was cloned into the pFuse vector without hIgG1 Fcfragment. The obtained expression vectors were confirmed by sequencing.FIGS. 8F and 8H provide depictions of a Moka1 peptide inserted into orreplacing at least a portion of the knob domain of a heavy chain regionof bovine BLV1H12 antibody.

Example 13 Expression and Purification of BLV1H12 Ab-Moka1 FusionAntibodies

BLV1H12-Moka1 fusion antibodies were expressed through transienttransfections of free style HEK 293 cells with vectors encodingBLV1H12-Moka1 fusion heavy chain and the BLV1H12 light chain.BLV1H12-Moka1 fusion antibodies were secreted into the culture mediumand harvested at 48 hours and 96 hours after transfection. TheBLV1H12-Moka1 fusion antibodies were purified by Protein A/Gchromatography (Thermo Fisher Scientific, IL), and analyzed by SDS-PAGEgel. FIG. 16A shows a SDS PAGE of the immunoglobulin fusion antibodiessAb-Moka1 L0 (n=0) and Ab-Moka1 L1 (n=1).

Example 14 In Vitro Study of BLV1H12-Moka1 Fusion Antibodies InhibitoryActivities on Human Peripheral Blood Mononuclear Cells (PBMCs)/T CellsActivation

Human PBMCs were isolated from fresh venous blood of healthy donorsthrough ficoll gradient centrifugation, followed by resuspension inRPMI1640 medium with 10% FBS and plating in 96-well plates at a densityof 1×10⁶ cells/mL. Human T cells were purified from the isolated PBMCsusing T cell enrichment kit. Purified PBMCs and T cells were pretreatedfor 1 h at 37° C. with 5% CO₂ with various concentrations of purifiedBLV1H12-Moka1 fusion antibodies and then activated by anti-CD3 and CD28antibodies. After 24 h treatment, supernatant was collected formeasurement of the levels of secreted TNF-α using ELISA kit. FIG. 17 andTable 13 shows BLV1H12-Moka1 fusion antibodies inhibitory activities onhuman peripheral blood mononuclear cells (PBMCs). FIG. 16B and Table 14shows BLV1H12-Moka1 fusion antibodies inhibitory activities on T cellsactivation.

TABLE 13 Concentration Ab Ab-Moka L0 Ab-Moka L1 (nM) F.I. F.I. F.I. 01966.657 1966.657 1966.657 2 2333.599333 1679.371333 1394.048 202186.372667 1441.220667 1294.799333 200 1981.540333 928.0533333773.3666667 400 1732.831333 664.9696667 505.102 N.A. 183.3106667183.3106667 183.3106667

TABLE 14 [Ab-Moka-L1] nM F.I. 0.3658 479.8675 1.09739 498.558 3.29218452.342 9.87654 445.013 29.62963 467.268 88.88889 360.2535 266.66667233.809 800 226.155

Example 15 Constructing Vectors of BLV1H12-VM24 Fusion Proteins forExpression in Mammalian Cells

A gene encoding Vm24 was synthesized by Genscript or IDT, and amplifiedby polymerase chain reaction (PCR). To optimize the folding andstability of BLV1H12-VM24 fusion proteins, flexible linkers of GGGGS orGGGSGGGGS were added on both ends of the Vm24 fragment. Subsequently,PCR fragments of VM24 with varied lengths of linkers were grafted intothe complementarity determining region 3 of the heavy chain (CDR3H) ofBLV1H12 antibody by exploiting overlap extension PCR, to replace the‘knob’ domain as shown in the crystal structure of BLV1H12. Theexpression vectors of BLV1H12-VM24 fusion proteins were generated byin-frame ligation of the amplified BLV1H12-VM24 fusion genes to thepFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly, the geneencoding the light chain of BLV1H12 antibody was cloned into the pFusevector without hIgG1 Fc fragment. The obtained expression vectors wereconfirmed by sequencing. FIGS. 8F, and 8H provide depictions of a VM24peptide inserted into or replacing at least a portion of the knob domainof a heavy chain region of bovine BLV1H12 antibody.

Example 16 Expression and Purification of BLV1H12-VM24 Fusion Antibodies

BLV1H12-VM24 fusion antibodies were expressed through transienttransfections of free style HEK 293 cells with vectors encodingBLV1H12-VM24 fusion heavy chain and the BLV1H12 light chain. ExpressedBLV1H12-VM24 fusion antibodies were secreted into the culture medium andharvested every 48 hours for twice after transfection. The BLV1H12-VM24fusion antibodies were purified by Protein A/G chromatography (ThermoFisher Scientific, IL), and analyzed by SDS-PAGE gel. FIG. 18A shows aSDS PAGE of the immunoglobulin constructs Ab-VM24 L1 (linker=GGGGS) andAb-VM24 L2 (first linker=GGGSGGGGS and second linker=GGGGSGGGS).

Example 17 In Vitro Study of BLV1H12-Vm24 Fusion Antibodies InhibitoryActivities on T Cells Activation

Human T cells were purified from the isolated PBMCs using T cellenrichment kit. Purified T cells were pretreated for 1 h at 37° C. with5% CO₂ with various concentrations of purified BLV1H12-VM24 fusionantibodies and then activated by anti-CD3 and CD28 antibodies. After 24h treatment, supernatant was collected for measurement of the levels ofsecreted TNF-a using ELISA kit. FIGS. 18B-C and Table 15 show theBLV1H12-VM24 fusion antibodies inhibitory activities on T cellsactivation.

TABLE 15 [Ab-VM24 L1] [Ab-VM24-L2] nM F.I. nM F.I. 0.45725 3427.0040.45725 3156.626 1.37174 3265.969 1.37174 3345.846 4.11523 3499.824.11523 3518.316 12.34568 3627.431 12.34568 3607.5755 37.03704 3575.481537.03704 3508.0475 111.11111 3085.439 111.11111 3220.2475 333.333331853.645 333.33333 2465.838 1000 990.818 1000 1306.0215

Example 18 Constructing Vectors of BLV1H12-Ex-4 Fusion Proteins forExpression in Mammalian Cells

A gene encoding Exendin-4 (Ex-4) was synthesized by Genscript or IDT,and amplified by polymerase chain reaction (PCR). A cleavage site ofFactor Xa was placed in front of the N-terminal of Exendin-4. Inaddition to this protease cleavage site, GGGGS linker followed with acysteine were also added on both ends of the Exendin-4 fragment.Subsequently, PCR fragments of Exendin-4 were grafted into thecomplementarity determining region 3 of the heavy chain (CDR3H) ofBLV1H12 antibody by exploiting overlap extension PCR, to replace the‘knob’ domain as shown in the crystal structure of BLV1H12. Theexpression vectors of BLV1H12-Ex-4 clip fusion proteins were generatedby in-frame ligation of the amplified BLV1H12-fusion genes to thepFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly, the geneencoding the light chain of BLV1H12 antibody was cloned into the pFusevector without hIgG1 Fc fragment. The obtained expression vectors wereconfirmed by sequencing. FIG. 8F, and 8I show cartoons depictingExendin-4 peptide inserted into or replacing at least a portion of theknob domain of an immunoglobulin heavy chain region. FIG. 8I alsodepicts the clipped version of the BLV1H12-Exendin-4 fusion protein,wherein Exendin-4 has a free N-terminus.

Example 19 Expression and Purification of BLV1H12-Ex-4 Clip FusionProteins

BLV1H12-Ex-4 fusion antibodies were expressed through transienttransfections of free style HEK 293 cells with vectors encodingBLV1H12-Ex-4 fusion heavy chain and the BLV1H12 light chain. ExpressedBLV1H12-Ex-4 fusion antibodies were secreted into the culture medium andharvested at 48 hours and 96 hours after transfection. The BLV1H12-Ex-4fusion antibodies were purified by Protein A/G chromatography (ThermoFisher Scientific, IL). BLV1H12-Ex-4 fusion antibodies were furthertreated with Factor Xa protease (GE Healthcare) following manufacture'sprotocol to release N-terminal of Ex-4 peptides fused to the BLV1H12antibody. After treatment, BLV1H12-Ex-4 fusion antibodies werere-purified by Protein A/G affinity column to remove protease andanalyzed by SDS-PAGE gel. FIG. 19 shows a western blot of expression ofthe immunoglobulin construct Ab-Exendin-4.

Example 20 In Vitro Study of BLV1H12-Ex-4 Clip Fusion AntibodiesActivation Activities on GLP-1 Receptor (GLP-1R)

HEK293 cells expressing surface GLP-1R and cAMP responsive luciferasereporter gene were seeded in 384 well plates at a density of 5000 cellsper well. After 24 h incubation at 37° C. with 5% CO₂, cells weretreated with various concentrations of Exendin-4 peptides andBLV1H12-Ex-4 clip fusion antibodies and incubated for another 24 h.Subsequently, luciferase assay was performed using One-Glo luciferasereagent according manufacture's instruction (Promega). FIG. 20 andTables 16-17 show the activity of Ab-Ex4 fusion antibodies on HEK293cells expressing GLP-1 receptor.

TABLE 16 Ab- Ab-GLP1 GLP1(RN) Ab (nM) RLU (nM) RLU Ab-Ex4 (nM) RLU (nM)RLU 1.26953 5200 1.26953 18600 1.26953 6120 1.26953 4740 2.53906 50002.53906 24500 2.53906 6360 2.53906 5800 5.07813 5600 5.07813 432005.07813 8500 5.07813 4400 10.15625 5500 10.15625 66560 10.15625 1042010.15625 5200 20.3125 5380 20.3125 105040 20.3125 19340 20.3125 778040.625 4600 40.625 143780 40.625 27960 40.625 13600 81.25 6140 81.25151060 81.25 54800 81.25 33760 162.5 5760 162.5 166640 162.5 90660 162.565800 325 5600 325 171400 325 117900 325 100920 650 5800 650 159780 650134760 650 140640 1300 14000 1300 184960 1300 159660 1300 169060

TABLE 17 Ab-Ex4(RN) Ex4 (nM) RLU (nM) RLU 0.00248 24120 0.00248 625000.00496 26320 0.00496 71840 0.00992 28140 0.00992 72160 0.01984 335000.01984 71360 0.03967 34180 0.03967 69720 0.07935 48860 0.07935 723800.15869 63460 0.15869 77680 0.31738 80740 0.31738 87220 0.63477 1172400.63477 93760 1.26953 128740 1.26953 134100 2.53906 153820 2.53906128120 5.07813 163020 5.07813 138220 10.15625 169360 10.15625 15870020.3125 161380 20.3125 165300 40.625 154920 40.625 175200 81.25 163700162.5 163860 325 164160 650 155700 1300 168740

Example 21 Constructing Vectors of BLV1H12-GLP-1 Clip Fusion Proteinsfor Expression in Mammalian Cells

A gene encoding GLP-1 was synthesized by Genscript or IDT, and amplifiedby polymerase chain reaction (PCR). To optimize the folding andstability of fusion proteins, flexible linkers of (GGGGS)n (n=0, 1) wereadded on both ends of the Moka1 fragment. Linkers of GGGGS or GGGSGGGGSwere added on both ends of the GLP-1 fragment. A cleavage site of FactorXa was placed in front of the N-terminal of GLP-1. In addition to thisprotease cleavage site, GGGGS linker followed with a cysteine were alsoadded on both ends of the GLP-1 fragment. Subsequently, PCR fragments ofGLP-1 were grafted into the complementarity determining region 3 of theheavy chain (CDR3H) of BLV1H12 antibody by exploiting overlap extensionPCR, to replace the ‘knob’ domain as shown in the crystal structure ofBLV1H12. The expression vectors of BLV1H12-GLP-1 clip fusion proteinswere generated by in-frame ligation of the amplified BLV1H12-GLP-1fusion genes to the pFuse-hIgG1-Fc backbone vector (InvivoGen, CA).Similarly, the gene encoding the light chain of BLV1H12 antibody wascloned into the pFuse vector without hIgG1 Fc fragment. The obtainedexpression vectors were confirmed by sequencing. FIGS. 8F and 8I showcartoons depicting a GLP1 peptide inserted into or replacing at least aportion of the knob domain of an immunoglobulin heavy chain region. FIG.8I also shows shows a clipped version of the BLV1H12-GLP-1 fusionprotein, wherein GLP-1 has a free N-terminus.

Example 22 Expression and Purification of BLV1H12-GLP-1 Clip FusionAntibodies

BLV1H12-GLP-1 fusion antibodies were expressed through transienttransfections of free style HEK 293 cells with vectors encodingBLV1H12-GLP-1 fusion heavy chain and the BLV1H12 light chain. ExpressedBLV1H12-GLP-1 fusion antibodies were secreted into the culture mediumand harvested at 48 hours and 96 hours after transfection. TheBLV1H12-GLP-1 fusion antibodies were purified by Protein A/Gchromatography (Thermo Fisher Scientific, IL). BLV1H12-GLP-1 fusionantibodies were further treated with Factor Xa protease (GE Healthcare)following manufacture's protocol to release N-terminal of GLP-1 peptidefused to the BLV1H12 antibody. After treatment, BLV1H12-GLP-1 clipfusion antibodies were re-purified by Protein A/G affinity column toremove protease and analyzed by SDS-PAGE gel. FIG. 19 provides a westernblot of expression of the immunoglobulin construct Ab-GLP-1.

Example 23 In Vitro Study of BLV1H12-GLP-1 Clip Fusion AntibodiesActivation Activities on GLP-1 Recepto (GLP-1R)

HEK293 cells expressing surface GLP-1R and cAMP responsive luciferasereporter gene were seeded in 384 well plates at a density of 5000 cellsper well. After 24 h incubation at 37° C. with 5% CO₂, cells weretreated with various concentrations of peptides and BLV1H12-GLP-1 clipfusion proteins and incubated for another 24 h. Subsequently, luciferaseassay was performed using One-Glo luciferase reagent accordingmanufacture's instruction (Promega). FIG. 20 and Table 16 show theactivity of Ab-GLP-1 f fusion antibodies on HEK293 cells expressingGLP-1 receptor.

Example 24 Constructing Vectors of BLV1H12-hEPO Fusion Proteins forExpression in Mammalian Cells

A gene encoding human EPO was synthesized by Genscript or IDT, andamplified by polymerase chain reaction (PCR). To optimize the foldingand stability of fusion proteins, flexible linkers of (GGGGS) were addedon both ends of human EPO. Subsequently, PCR fragments of hEPO weregrafted into the complementarity determining region 3 of the heavy chain(CDR3H) of BLV1H12 antibody by exploiting overlap extension PCR, toreplace the ‘knob’ domain as shown in the crystal structure of BLV1H12.The expression vectors of BLV1H12-hEPO fusion proteins were generated byin-frame ligation of the amplified BLV1H12-fusion genes to thepFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly, the geneencoding the light chain of BLV1H12 antibody was cloned into the pFusevector without hIgG1 Fc fragment. The obtained expression vectors wereconfirmed by sequencing. FIGS. 8F and 8J show cartoons depicting hEPOpeptide attached to the knob domain of an immunoglobulin heavy chainregion.

Example 25 Expression and Purification of BLV1H12-hEPO Fusion Antibodies

BLV1H12-hEPO fusion antibodies were expressed through transienttransfections of free style HEK 293 cells with vectors encodingBLV1H12-hEPO fusion heavy chain and the BLV1H12 light chain. ExpressedBLV1H12-hEPO fusion antibodies were secreted into the culture medium andharvested at 48 hours and 96 hours after transfection. The BLV1H12-hEPOfusion antibodies were purified by Protein A/G chromatography (ThermoFisher Scientific, IL), and analyzed by SDS-PAGE gel.

Example 26 In Vitro Study of BLV1H12-hEPO Fusion Antibody ProliferativeActivities on TF-1 Cells

TF-1 cells were obtained from American Type Culture Collection (ATCC),VA, and cultured in RPMI-1640 medium supplemented with 10% fetal bovineserum (FBS), penicillin, streptomycin and 2 ng/mL granulocyte-macrophagecolony-stimulating factor (GM-CSF). For proliferation assay, TF-1 cellswere washed three times with RPMI-1640 medium and resuspended inRPMI-1640 medium with 10% FBS and penicillin and streptomycin at adensity of 1.5×10⁵ cells/ml. Cells were plated in 96-well plates andtreated with varied concentrations of BLV1H12-hEPO fusion antibodies.After 72 h of incubation at 37° C. with 5% CO₂, cells viabilities weremeasured using AlamarBlue (Invitrogen) assay following manufacture'sinstruction. FIG. 21 and Table 18 show the proliferative activity ofAb-hEPO fusion antibodies on TF1 cells.

TABLE 18 hEPO Ab-hEPO Ab (nM) F.I. (nM) F.I. (nM) F.I. 2.25E−05 4261.3649.42E−05 4361.92 9.42E−05 4108.119 1.13E−04 4722.771 4.71E−04 4324.0374.71E−04 4223.257 5.63E−04 4481.459 0.00236 4198.65 0.00236 4274.070.00282 5128.302 0.01178 4757.196 0.01178 4267.586 0.01408 5522.4590.05888 5265.069 0.05888 3905.529 0.0704 7125.093 0.2944 6430.723 0.29444091.452 0.352 8629.194 1.472 8963.889 1.472 4109.106 1.76 9748.017 7.3610330.18 7.36 4071.234 8.8 10392.97 36.8 10776.73 36.8 4100.011 449357.346 184 10330.44 184 4413.497

Example 27 Constructing Vectors of BLV1H12-hFGF21 Fusion Proteins forExpression in Mammalian Cells

A gene encoding human FGF21 (hFGF21) was synthesized by Genscript orIDT, and amplified by polymerase chain reaction (PCR). To optimize thefolding and stability of fusion proteins, flexible linkers of (GGGGS)were added on both ends of human FGF21. Subsequently, PCR fragments ofhFGF21 were grafted into the complementarity determining region 3 of theheavy chain (CDR3H) of BLV1H12 antibody by exploiting overlap extensionPCR, to replace the ‘knob’ domain as shown in the crystal structure ofBLV1H12. The expression vectors of BLV1H12-hFGF21 fusion proteins weregenerated by in-frame ligation of the amplified BLV1H12-fusion genes tothe pFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly, the geneencoding the light chain of BLV1H12 antibody was cloned into the pFusevector without hIgG1 Fc fragment. The obtained expression vectors wereconfirmed by sequencing.

Example 28 Expression and Purification of BLV1H12-hFGF21 Fusion Antibody

BLV1H12-hFGF21 fusion antibodies were expressed through transienttransfections of free style HEK 293 cells with vectors encodingBLV1H12-hFGF21 fusion heavy chain and the BLV1H12 light chain. ExpressedBLV1H12-hFGF21 fusion antibodies were secreted into the culture mediumand harvested every 48 hours for twice after transfection. TheBLV1H12-hFGF21 fusion antibodies were purified by Protein A/Gchromatography (Thermo Fisher Scientific, IL), and analyzed by SDS-PAGEgel.

Example 29 Constructing Vectors of BLV1H12-hGMCSF Fusion Proteins forExpression in Mammalian Cells

A gene encoding human GMCSF (hGMCSF) was synthesized by Genscript orIDT, and amplified by polymerase chain reaction (PCR). To optimize thefolding and stability of fusion proteins, flexible linkers of (GGGGS)were added on both ends of human GMCSF. Subsequently, PCR fragments ofhGMCSF were grafted into the complementarity determining region 3 of theheavy chain (CDR3H) of BLV1H12 antibody by exploiting overlap extensionPCR, to replace the ‘knob’ domain as shown in the crystal structure ofBLV1H12. The expression vectors of BLV1H12-hGMCSF fusion proteins weregenerated by in-frame ligation of the amplified BLV1H12-hGMCSF fusiongenes to the pFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly,the gene encoding the light chain of BLV1H12 antibody was cloned intothe pFuse vector without hIgG1 Fc fragment. The obtained expressionvectors were confirmed by sequencing.

Example 30 Expression and Purification of BLV1H12-hGMCSF FusionAntibodies

BLV1H12-hGMCSF fusion antibodies can be expressed through transienttransfections of free style HEK 293 cells with vectors encodingBLV1H12-hGMCSF fusion heavy chain and the BLV1H12 light chain. ExpressedBLV1H12-hGMCSF fusion antibodies can be secreted into the culture mediumand harvested at 48 hours and 96 hours after transfection. TheBLV1H12-hGMCSF fusion antibodies can be purified by Protein A/Gchromatography (Thermo Fisher Scientific, IL), and analyzed by SDS-PAGEgel.

Example 31 Constructing Vectors of BLV1H12-hIFN-b Proteins forExpression in Mammalian Cells

A gene encoding human interferon-beta (hIFN-b) was synthesized byGenscript or IDT, and amplified by polymerase chain reaction (PCR). Tooptimize the folding and stability of fusion proteins, flexible linkersof (GGGGS) were added on both ends of human interferon-beta.Subsequently, PCR fragments of hIFN-b were grafted into thecomplementarity determining region 3 of the heavy chain (CDR3H) ofBLV1H12 antibody by exploiting overlap extension PCR, to replace the‘knob’ domain as shown in the crystal structure of BLV1H12. Theexpression vectors of BLV1H12-hIFN-b fusion proteins were generated byin-frame ligation of the amplified BLV1H12-hIFN-b fusion genes to thepFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly, the geneencoding the light chain of BLV1H12 antibody was cloned into the pFusevector without hIgG1 Fc fragment. The obtained expression vectors wereconfirmed by sequencing.

Example 32 Expression and Purification of BLV1H12-hIFN-b FusionAntibodies

BLV1H12-hIFN-b fusion antibodies can be expressed through transienttransfections of free style HEK 293 cells with vectors encodingBLV1H12-hIFN-b fusion heavy chain and the BLV1H12 light chain. ExpressedBLV1H12-hIFN-b fusion antibodies can be secreted into the culture mediumand harvested at 48 hours and 96 hours after transfection. TheBLV1H12-hIFN-b fusion antibodies can be purified by Protein A/Gchromatography (Thermo Fisher Scientific, IL), and analyzed by SDS-PAGEgel.

Example 33 Constructing Vectors of BLV1H12-Fusion Proteins forExpression in Mammalian Cells

Genes encoding various genes were synthesized by Genscript (NJ, USA) andamplified by polymerase chain reaction (PCR). To optimize the foldingand stability of the immunoglobulin constructs, one or more flexiblelinkers of (GGGGS)n (n=0, 1), GGGSGGGGS, and/or GGGGSGGGS were added onboth ends of the gene fragment. Subsequently, PCR fragments of the geneswith varied lengths of linkers were grafted into the complementaritydetermining region 3 of the heavy chain (CDR3H) of BLV1H12 antibody byexploiting overlap extension PCR, to replace at least a portion of the‘knob’ domain as shown in the crystal structure of BLV1H12 (FIGS.8A-8J). The expression vectors of BLV1H12-fusion proteins were generatedby in-frame ligation of the amplified BLV1H12-fusion genes to thepFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly, the geneencoding the light chain of BLV1H12 antibody was cloned into the pFusevector without hIgG1 Fc fragment. The obtained expression vectors wereconfirmed by sequencing.

Nucleic acid sequences of the BLV1H12-fusion proteins are displayed inTable 19 (SEQ ID NOS: 1-15). Peptide sequences of the BLVH12-fusionproteins are displayed in Table 21 (SEQ ID NOS: 23-37). As shown in theTable 19 and Table 21, the bovine heavy chain sequence is in bold font;the human heavy chain sequence is highlighted with a dashed underline;the non-antibody sequence is in italicized font; the stalk domain is inbold font and underlined; the knob domain is in bold font and doubleunderlined; the linker sequence is in italicized font and squigglyunderlined.

Example 34 Constructing Vectors of BLV1H12-IL8 Fusion Proteins forExpression in Mammalian Cells

Gene encoding a human IL-8 sequence (see, e.g., SEQ ID NO: 317corresponding to amino acids 26-99 of IL-8, designated IL8 herein) wasamplified from cDNA (OriGene) by polymerase chain reaction (PCR). Insome constructs, a linker (e.g., GSG or repeats of GSG) may be added onone or both ends of the IL8 fragment. Subsequently, PCR fragments of IL8with or without a linker were grafted into the complementaritydetermining region 3 of the heavy chain (CDR3H) of BLV1H12 antibody byPCR, to replace the ‘knob’ domain as shown in the crystal structure ofBLV1H12. The expression vectors of BLV1H12-IL8 fusion proteins weregenerated by in-frame ligation of the amplified BLV1H12-IL8 fusion genesto CH1—CH2-CH3 in a pFuse-backbone vector (InvivoGen, CA). TheBLV1H12-IL8 heavy chain variable region sequence is shown as SEQ ID NO:16 (nucleotide) and SEQ ID NO: 38 (amino acid). Similarly, the geneencoding the light chain of BLV1H12 antibody was cloned into a pFusevector without hIgG1 Fc fragment. The obtained expression vectors wereconfirmed by sequencing.

Example 35 Expression of BLV1H12-IL8 Fusion Proteins

BLV1H12-IL8 fusion proteins were expressed through transienttransfections of 293T cells or Freestyle™ 293-F cells with vectorsencoding BLV1H12-IL8 heavy and light chains. Expressed fusion proteinswere secreted into the culture medium and culture supernatants obtainedafter 2 days of cell culture. The expression of BLV1H12-IL8 fusionproteins in the supernatants was determined by ELISA to be 37.2 nM

Example 36 In Vitro Study of Activities of the BLV1H12-IL8 FusionProteins on CXCR1 Expressing Cells

Briefly, a cell line expressing functionally validated CXCR1 derivedfrom U2OS cells was obtained from DiscoveRx and cultured permanufacturer's instructions (Cat#93-0226C3, DiscoveRx Corporation,Freemont, Calif.). The parental cell line U20S was obtained from ATCCand cultured under the same conditions as the CXCR1 cells. Cell culturesupernatants from Example 2 above were then tested for binding to cellsby flow cytometry. The adherent U20S or CXCR1-U20S cells weredissociated with Accutase (Innovative Cell Technologies, Inc., SanDiego, Calif.), neutralized with an equal volume of media containing 10%serum, centrifuged at 1000 g, and resuspended in PBS with 2% BSA. Next,cells were dispensed into microtiter plates to achieve between 30,000 to300,000 cells per well, centrifuged again, and resuspended in cellculture supernatant containing expressed IgG, or a dilution ofIgG-containing cell culture supernatant. A fluorescent-conjugatedanti-Human Fc antibody was used to detect binding of theexpressedBLV1H12-IL8 fusion proteins to cells. Subsequently, cellfluorescence was measured by flow cytometry (e.g., FACS), and medianArbitrary Fluorescence Units (AFU) were calculated, revealing the extentof IgG binding to those cells. The ratio of median fluorescence (IgGbinding) of CXCR1-U20S cells versus U20S parental cells shows that theBLV1H12-IL8 fusion protein has specificity for CXCR1 (Table 27).

TABLE 27 BLV1H12 (Median BLV1H12-IL8 (Median Arbitrary FluorescenceArbitrary Fluorescence Units (AFU)) Units (AFU)) Parental U2OS 4 76CXCR1-U2OS 4 707

Example 37 Constructing Vectors of BLV1H12-Ziconotide Fusion Proteinsfor Expression in Mammalian Cells

Gene encoding a ziconotide sequence (see, e.g., SEQ ID NO: 318) wasprepared from multiple oligonucleotides and then amplified by polymerasechain reaction (PCR). In some constructs, a linker (e.g., GSG or repeatsof GSG) may be added on one or both ends of the ziconotide fragment.Subsequently, PCR fragments of ziconotide with or without a linker weregrafted into the complementarity determining region 3 of the heavy chain(CDR3H) of BLV1H12 antibody by PCR, to replace the ‘knob’ domain asshown in the crystal structure of BLV1H12. The expression vectors ofBLV1H12-ziconotide fusion proteins were generated by in-frame ligationof the amplified BLV1H12-ziconotide fusion genes to CH1-CH2-CH3 in apFuse-backbone vector (InvivoGen, CA). The BLV1H12-ziconotide heavychain variable region sequence is shown as SEQ ID NO: 17 (nucleotide)and SEQ ID NO: 39 (amino acid). Similarly, the gene encoding the lightchain of BLV1H12 antibody was cloned into a pFuse vector without hIgG1Fc fragment. The obtained expression vectors were confirmed bysequencing.

Example 38 Expression of BLV1H12-Ziconotide Fusion Proteins

BLV1H12-ziconotide fusion proteins were expressed through transienttransfections of 293T cells or Freestyle™ 293-F cells with vectorsencoding BLV1H12-ziconotide heavy and light chains. Expressed fusionproteins were secreted into the culture medium and culture supernatantsobtained after 2 days of cell culture. The expression ofBLV1H12-ziconotide fusion proteins in the supernatants was determined byELISA and normalized as compared to the IL8 construct in Example 35above to be 94.7% of the IL8 construct.

Example 39 Constructing Vectors of BLV1H12-Somatostatin Fusion Proteinsfor Expression in Mammalian Cells

Gene encoding a somatostatin sequence (see, e.g., SEQ ID NO: 319) wasprepared from multiple oligonucleotides and then amplified by polymerasechain reaction (PCR). In some constructs, a linker (e.g., GSG or repeatsof GSG) may be added on one or both ends of the somatostatin fragment.Subsequently, PCR fragments of somatostatin with or without a linkerwere grafted into the complementarity determining region 3 of the heavychain (CDR3H) of BLV1H12 antibody by PCR, to replace the ‘knob’ domainas shown in the crystal structure of BLV1H12. The expression vectors ofBLV1H12-somatostatin fusion proteins were generated by in-frame ligationof the amplified BLV1H12-somatostatin fusion genes to CH1-CH2-CH3 in apFuse-backbone vector (InvivoGen, CA). The BLV1H12-somatostatin heavychain variable region sequence is shown as SEQ ID NO: 18 (nucleotide)and SEQ ID NO: 40 (amino acid). Similarly, the gene encoding the lightchain of BLV1H12 antibody was cloned into a pFuse vector without hIgG1Fc fragment. The obtained expression vectors were confirmed bysequencing.

Example 40 Expression of BLV1H12-Somatostatin Fusion Proteins

BLV1H12-somatostatin fusion proteins were expressed through transienttransfections of 293T cells or Freestyle™ 293-F cells with vectorsencoding BLV1H12-somatostatin heavy and light chains. Expressed fusionproteins were secreted into the culture medium and culture supernatantsobtained after 2 days of cell culture. The expression ofBLV1H12-somatostatin fusion proteins in the supernatants was determinedby ELISA and normalized as compared to the IL8 construct in Example 35above to be 46.5% of the IL8 construct.

Example 41 Constructing Vectors of BLV1H12-Chlorotoxin Fusion Proteinsfor Expression in Mammalian Cells

Gene encoding a chlorotoxin sequence (see, e.g., SEQ ID NO: 320) wasprepared from multiple oligonucleotides and then amplified by polymerasechain reaction (PCR). In some constructs, a linker (e.g., GSG or repeatsof GSG) may be added on one or both ends of the chlorotoxin fragment.Subsequently, PCR fragments of chlorotoxin with or without a linker weregrafted into the complementarity determining region 3 of the heavy chain(CDR3H) of BLV1H12 antibody by PCR, to replace the ‘knob’ domain asshown in the crystal structure of BLV1H12. The expression vectors ofBLV1H12-chlorotoxin fusion proteins were generated by in-frame ligationof the amplified BLV1H12-chlorotoxin fusion genes to CH1-CH2-CH3 in apFuse-backbone vector (InvivoGen, CA). The BLV1H12-chlorotoxin heavychain variable region sequence is shown as SEQ ID NO: 19 (nucleotide)and SEQ ID NO: 41 (amino acid). Similarly, the gene encoding the lightchain of BLV1H12 antibody was cloned into a pFuse vector without hIgG1Fc fragment. The obtained expression vectors were confirmed bysequencing.

Example 42 Expression of BLV1H12-Chlorotoxin Fusion Proteins

BLV1H12-chlorotoxin fusion proteins were expressed through transienttransfections of 293T cells or Freestyle™ 293-F cells with vectorsencoding BLV1H12-chlorotoxin heavy and light chains. Expressed fusionproteins were secreted into the culture medium and culture supernatantsobtained after 2 days of cell culture. The expression ofBLV1H12-chlorotoxin fusion proteins in the supernatants was determinedby ELISA and normalized as compared to the IL8 construct in Example 35above to be 39.7% of the IL8 construct.

Example 43 Constructing Vectors of BLV1H12-SDF-1 (Alpha) Fusion Proteinsfor Expression in Mammalian Cells

Gene encoding a SDF-1 alpha sequence (see, e.g., SEQ ID NO: 321) wasamplified from cDNA (OriGene) by polymerase chain reaction (PCR). Insome constructs, a linker (e.g., GSG or repeats of GSG) may be added onone or both ends of the SDF-1 (alpha) fragment. Subsequently, PCRfragments of SDF-1 (alpha) with or without a linker were grafted intothe complementarity determining region 3 of the heavy chain (CDR3H) ofBLV1H12 antibody by PCR, to replace the ‘knob’ domain as shown in thecrystal structure of BLV1H12. The expression vectors of BLV1H12-SDF-1(alpha) fusion proteins were generated by in-frame ligation of theamplified BLV1H12-SDF-1 (alpha) fusion genes to CH1-CH2-CH3 in apFuse-backbone vector (InvivoGen, CA). The BLV1H12-SDF-1 (alpha) heavychain variable region sequence is shown as SEQ ID NO: 20 (nucleotide)and SEQ ID NO: 42 (amino acid). Similarly, the gene encoding the lightchain of BLV1H12 antibody was cloned into a pFuse vector without hIgG1Fc fragment. The obtained expression vectors were confirmed bysequencing.

Example 44 Expression of BLV1H12-SDF-1 (Alpha) Fusion Proteins

BLV1H12-SDF-1 (alpha) fusion proteins were expressed through transienttransfections of 293T cells or Freestyle™ 293-F cells with vectorsencoding BLV1H12-SDF-1 (alpha) heavy and light chains. Expressed fusionproteins were secreted into the culture medium and culture supernatantsobtained after 2 days of cell culture. The expression of BLV1H12-SDF-1(alpha) fusion proteins in the supernatants was determined by ELISA andnormalized as compared to the IL8 construct in Example 35 above to be38.9% of the IL8 construct.

Example 45 Constructing Vectors of BLV1H12-IL21 Fusion Proteins forExpression in Mammalian Cells

Gene encoding an IL21 sequence (see, e.g., SEQ ID NO: 322) was amplifiedfrom cDNA (OriGene) by polymerase chain reaction (PCR). In someconstructs, a linker (e.g., GSG or repeats of GSG) may be added on oneor both ends of the IL21 fragment. Subsequently, PCR fragments of IL21with or without a linker were grafted into the complementaritydetermining region 3 of the heavy chain (CDR3H) of BLV1H12 antibody byPCR, to replace the ‘knob’ domain as shown in the crystal structure ofBLV1H12. The expression vectors of BLV1H12-IL21 fusion proteins weregenerated by in-frame ligation of the amplified BLV1H12-IL21 fusiongenes to CH1-CH2-CH3 in a pFuse-backbone vector (InvivoGen, CA). TheBLV1H12-IL21 heavy chain variable region sequence is shown as SEQ ID NO:21 (nucleotide) and SEQ ID NO: 43 (amino acid). Similarly, the geneencoding the light chain of BLV1H12 antibody was cloned into a pFusevector without hIgG1 Fc fragment. The obtained expression vectors wereconfirmed by sequencing.

Example 46 Expression of BLV1H12-IL21 Fusion Proteins

BLV1H12-IL21 fusion proteins were expressed through transienttransfections of 293T cells or Freestyle™ 293-F cells with vectorsencoding BLV1H12-IL21 heavy and light chains. Expressed fusion proteinswere secreted into the culture medium and culture supernatants obtainedafter 2 days of cell culture. The expression of BLV1H12-IL21 fusionproteins in the supernatants was determined by ELISA and normalized ascompared to the IL8 construct in Example 35 above to be 32.3% of the IL8construct.

Example 47 Constructing Vectors of BLV1H12-Protoxin2 Fusion Proteins forExpression in Mammalian Cells

Gene encoding protoxin2 was synthesized by Genscript or IDT, andamplified by polymerase chain reaction (PCR). To optimize the foldingand stability of fusion proteins, flexible linkers of GGGGS were addedon both ends of protoxin2 fragments. Subsequently, PCR fragments ofprotoxin2 (called protoxin2-L1) were grafted into the complementaritydetermining region 3 of the heavy chain (CDR3H) of BLV1H12 antibody byexploiting overlap extension PCR, to replace the ‘knob’ domain as shownin the crystal structure of BLV1H12. The expression vectors ofBLV1H12-fusion proteins were generated by in-frame ligation of theamplified BLV1H12-protoxin2-L1 fusion genes (SEQ ID NO: 15) to thepFuse-hIgG1-Fc backbone vector (InvivoGen, CA). Similarly, the geneencoding the light chain of BLV1H12 antibody was cloned into the pFusevector without hIgG1 Fc fragment. The obtained expression vectors wereconfirmed by sequencing.

Example 48 Expression and Purification of BLV1H12-Protoxin2-L1 FusionProteins

BLV1H12-protoxin2-L1 fusion antibodies were expressed through transienttransfections of free style HEK 293 cells with vectors encodingBLV1H12-protoxin2 fusion heavy chain and the BLV1H12 light chain.Expressed BLV1H12-protoxin2-L1 fusion antibodies were secreted into theculture medium and harvested at 48 hours and 96 hours aftertransfection. The BLV1H12-protoxin2-L1 fusion antibodies were purifiedby Protein A/G chromatography (Thermo Fisher Scientific, IL), andanalyzed by SDS-PAGE gel (FIG. 15B).

Example 49 Constructing Vectors of BLV1H12-ProTxII Fusion Proteins forExpression in Mammalian Cells

Gene encoding a ProTxII sequence (see, e.g., SEQ ID NO: 323) wasprepared from multiple oligonucleotides and then amplified by polymerasechain reaction (PCR). In some constructs, a linker (e.g., GSG or repeatsof GSG) may be added on one or both ends of the ProTxII fragment.Subsequently, PCR fragments of ProTxII with or without a linker weregrafted into the complementarity determining region 3 of the heavy chain(CDR3H) of BLV1H12 antibody by PCR, to replace the ‘knob’ domain asshown in the crystal structure of BLV1H12. The expression vectors ofBLV1H12-ProTxII fusion proteins were generated by in-frame ligation ofthe amplified BLV1H12-ProTxII fusion genes to CH1-CH2-CH3 in apFuse-backbone vector (InvivoGen, CA). The BLV1H12-ProTxII heavy chainvariable region sequence is shown as SEQ ID NO: 22 (nucleotide) and SEQID NO: 44 (amino acid). Similarly, the gene encoding the light chain ofBLV1H12 antibody was cloned into a pFuse vector without hIgG1 Fcfragment. The obtained expression vectors were confirmed by sequencing.

Example 50 Expression of BLV1H12-ProTxII Fusion Proteins

BLV1H12-ProTxII fusion proteins were expressed through transienttransfections of 293T cells or Freestyle™ 293-F cells with vectorsencoding BLV1H12-ProTxII heavy and light chains. Expressed fusionproteins were secreted into the culture medium and culture supernatantsobtained after 2 days of cell culture. The expression of BLV1H12-ProTxIIfusion proteins in the supernatants was determined by ELISA andnormalized as compared to the IL8 construct in Example 35 above to be2.1% of the IL8 construct.

For the disclosure herein, the very least, and not as an attempt tolimit the application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of the disclosure areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

TABLE 19 SEQ ID Description NO: Sequence Light Chain 1CAGGCCGTCCTGAACCAGCCAAGCAGCGTCTCCGGGTCTCTGGGGCAGCGGGTCTCAATCACCTGTAGCGGGTCTTCCTCCAATGTCGGCAACGGCTACGTGTCTTGGTATCAGCTGATCCCTGGCAGTGCCCCACGAACCCTGATCTACGGCGACACATCCAGAGCTTCTGGGGTCCCCGATCGGTTCTCAGGGAGCAGATCCGGAAACACAGCTACTCTGACCATCAGCTCCCTGCAGGCTGAGGACGAAGCAGATTATTTCTGCGCATCTGCCGAGGACTCTAGTTCAAATGCCGTGTTTGGAAGCGGCACCACACTGACAGTCCTGGGGCAGCCCAAGAGTCCCCCTTCAGTGACTCTGTTCCCACCCTCTACCGAGGAACTGAACGGAAACAAGGCCACACTGGTGTGTCTGATCAGCGACTTTTACCCTGGATCCGTCACTGTGGTCTGGAAGGCAGATGGCAGCACAATTACTAGGAACGTGGAAACTACCCGCGCCTCCAAGCAGTCTAATAGTAAATACGCCGCCAGCTCCTATCTGAGCCTGACCTCTAGTGATTGGAAGTCCAAAGGGTCATATAGCTGCGAAGTGACCCATGAAGGCTCAACCGTGACTAAGACTGT GAAACCATCCGAGTGCTCC Heavy Chain-2 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCA no insertionAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ACCAGGAAACTAAGAAATACCAG AGCTGTCCTGACGGCTATCGGGAGAGATCTGATTGCAGTAATAGGCCAGCTTGTGGCACATCCGACTGCTGTCGCGTGTCTGTCTTCGGGAACTGCCTGACTACCCTGCCTGTGTCCTACTCT TAT ACCTACAATTATGAATGGCATGTGGATGTCTGGGGAC AGGGCCTGCTGGTGACAGTCTCTAGT IFN-beta 3CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC

AATTTTCAGTGTCAGAAGCTCCTGTGGCAATTGAATGGGAGGCTTGAATACTGCCTCAAGGACAGGATGAACTTTGACATCCCTGAGGAGATTAAGCAGCTGCAGCAGTTCCAGAAGGAGGACGCCGCATTGACCATCTATGAGATGCTCCAGAACATCTTTGCTATTTTCAGACAAGATTCATCTAGCACTGGCTGGAATGAGACTATTGTTGAGAACCTCCTGGCTAATGTCTATCATCAGATAAACCATCTGAAGACAGTCCTGGAAGAAAAACTGGAGAAAGAAGATTTCACCAGGGGAAAACTCATGAGCAGTCTGCACCTGAAAAGATATTATGGGAGGATTCTGCATTACCTGAAGGCCAAGGAGTACAGTCACTGTGCCTGGACCATAGTCAGAGTGGAAATCCTAAGGAACTT

GGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAA

bGCSF-L0 4 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ACCAGGAAACTAAGAAATACCAG AGC ACCCCCCTTGGCCCTGCCCGATCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAAATCCAGGCTGATGGCGCCGAGCTGCAGGAGAGGCTGTGTGCCGCCCACAAGCTGTGCCACCCGGAGGAGCTGATGCTGCTCAGGCACTCTCTGGGCATCCCCCAGGCTCCCCTAAGCAGCTGCTCCAGCCAGTCCCTGCAGCTGACGAGCTGCCTGAACCAACTACACGGCGGCCTCTTTCTCTACCAGGGCCTCCTGCAGGCCCTGGCGGGCATCTCCCCAGAGCTGGCCCCCACCTTGGACACACTGCAGCTGGACGTCACTGACTTTGCCACGAACATCTGGCTGCAGATGGAGGACCTGGGGGCGGCCCCCGCTGTGCAGCCCACCCAGGGCGCCATGCCGACCTTCACTTCAGCCTTCCAACGCAGAGCAGGAGGGGTCCTGGTTGCTTCCCAGCTGCATCGTTTCCTGGAGCTGGCATACCGTGGCCTGCG CTACCTTGCTGAGCCC TCTTATACCTACAATTATGAATG G CATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGC

bGCSF-L1 5 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC

CTGCTCAAGTGCTTAGAGCAAGTGAGGAAAATCCAGGCTGATGGCGCCGAGCTGCAGGAGAGGCTGTGTGCCGCCCACAAGCTGTGCCACCCGGAGGAGCTGATGCTGCTCAGGCACTCTCTGGGCATCCCCCAGGCTCCCCTAAGCAGCTGCTCCAGCCAGTCCCTGCAGCTGACGAGCTGCCTGAACCAACTACACGGCGGCCTCTTTCTCTACCAGGGCCTCCTGCAGGCCCTGGCGGGCATCTCCCCAGAGCTGGCCCCCACCTTGGACACACTGCAGCTGGACGTCACTGACTTTGCCACGAACATCTGGCTGCAGATGGAGGACCTGGGGGCGGCCCCCGCTGTGCAGCCCACCCAGGGCGCCATGCCGACCTTCACTTCAGCCTTCCAACGCAGAGCAGGAGGGGTCCTGGTTGCTTCCCAGCTGCATCGTTTCCTGGAGCTGGCA

TCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCA

GMCSF 6 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC

GGGAGCATGTGAATGCCATCCAGGAGGCCCGGCGTCTCCTGAACCTGAGTAGAGACACTGCTGCTGAGATGAATGAAACAGTAGAAGTCATCTCAGAAATGTTTGACCTCCAGGAGCCGACCTGCCTACAGACCCGCCTGGAGCTGTACAAGCAGGGCCTGCGGGGCAGCCTCACCAAGCTCAAGGGCCCCTTGACCATGATGGCCAGCCACTACAAGCAGCACTGCCCTCCAACCCCGGAAACTTCCTGTGCAACCCAGATTATCACCTTTGAAAGTTTCAAAGAGAACCTGAAGGACTTTCTGCTTGTCATCCCCTTTGACTGCTGGGAGCC

TTATGAATGG CATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGT

hFGF21 7 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC

GGGCCAAGTCCGGCAGCGGTACCTCTACACAGATGATGCCCAGCAGACAGAAGCCCACCTGGAGATCAGGGAGGATGGGACGGTGGGGGGCGCTGCTGACCAGAGCCCCGAAAGTCTCCTGCAGCTGAAAGCCTTGAAGCCGGGAGTTATTCAAATCTTGGGAGTCAAGACATCCAGGTTCCTGTGCCAGCGGCCAGATGGGGCCCTGTATGGATCGCTCCACTTTGACCCTGAGGCCTGCAGCTTCCGGGAGCTGCTTCTTGAGGACGGATACAATGTTTACCAGTCCGAAGCCCACGGCCTCCCGCTGCACCTGCCAGGGAACAAGTCCCCACACCGGGACCCTGCACCCCGAGGACCAGCTCGCTTCCTGCCACTACCAGGCCTGCCCCCCGCACCCCCGGAGCCACCCGGAATCCTGGCCCCCCAGCCCCCCGATGTGGGCTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCA

TTATGAATGG CATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGT

Ex-4 8 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC

TGCTCTTATACCTACAATTATGAATGG CATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAA

hGLP-1 9 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC

TGCTCT TATACCTACAATTATGAATGG CATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAA

hEPO 10 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC

AGAGGTACCTCTTGGAGGCCAAGGAGGCCGAGAATATCACGACGGGCTGTGCTGAACACTGCAGCTTGAATGAGAATATCACTGTCCCAGACACCAAAGTTAATTTCTATGCCTGGAAGAGGATGGAGGTCGGGCAGCAGGCCGTAGAAGTCTGGCAGGGCCTGGCCCTGCTGTCGGAAGCTGTCCTGCGGGGCCAGGCCCTGTTGGTCAACTCTTCCCAGCCGTGGGAGCCCCTGCAGCTGCATGTGGATAAAGCCGTCAGTGGCCTTCGCAGCCTCACCACTCTGCTTCGGGCTCTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCAGATGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACACTTTCCGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGAAAGCTGAAGCTGTACACAGGGGAGGCCTGCAGGACAGGGG

AATGG CATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACA

Moka-L0 11 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC ACCAGGAAACTAAGAAATACCAG AGC ATCAACGTGAAGTGCAGCCTGCCCCAGCAGTGCATCAAGCCCTGCAAGGACGCCGGCATGCGGTTCGGCAAGTGCATGAACAAGAAGTGCAGGTG CTACAGC TCTTATACCTACAATTATGAATGG CATGTGG ATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAAC

Moka-L1 12 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC

ATACCTACAATTATGAATGG CATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCAAATCTTG

VM-24-L1 13 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC

TCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTGGACAAAGCAGTGGAACCCA

VM-24-L2 14 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC

AATTATGAATGG CATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAA

Protoxin2-L1 15 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACC ACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGC

TATGAATGG CATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTCTCTAGTGCTTCCACAACTGCACCAAAGGTGTACCCCCTGTCAAGCTGCTGTGGGGACAAATCCTCTAGTACCGTGACACTGGGATGCCTGGTCTCAAGCTATATGCCCGAGCCTGTGACTGTCACCTGGAACTCAGGAGCCCTGAAAAGCGGAGTGCACACCTTCCCAGCTGTGCTGCAGTCCTCTGGCCTGTATAGCCTGAGTTCAATGGTGACAGTCCCCGGCAGTACTTCAGGGCAGACCTTCACCTGTAATGTGGCCCATCCTGCCAGCTCCACCAAAGTG

TABLE 20  SEQ ID Description NO: Sequence BLV1H12-IL8 16CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAAGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCCCAAGGAGTGCTAAAGAACTTAGATGTCAGTGCATAAAGACATACTCCAAACCTTTCCACCCCAAGTTCATCAAGGAGCTGAGAGTGATTGAGAGTGGACCACACTGCGCCAACACAGAGATTATTGTAAAGCTTTCTGATGGGAGAGAGCTCTGCCTGGACCCCAAGGAAAACTGGGTGCAGAGGGTCGTGGAGAAGTTCTTGAAGAGGGCTGAGAACTCAGGCAGCGGTTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTC BLV1H12- 17CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA ZiconotideGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCTGCAAGGGCAAAGGTGCGAAATGCAGCCGCCTGATGTATGATTGCTGTACCGGGTCCTGCCGCAGTGGCAAGTGCTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACAGTC BLV1H12- 18CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA SomatostatinGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCGCTGGCTGCAAGAATTTCTTCTGGAAGACTTTCACATCCTGTGGTTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGCTGGTGACA GTC BLV1H12- 19CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA ChlorotoxinGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCATGTGTATGCCCTGCTTCACGACCGATCACCAGATGGCGCGCAAATGCGATGACTGTTGCGGCGGTAAAGGTCGCGGAAAGTGCTATGGCCCGCAGTGTCTGTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGG ACAGGGCCTGCTGGTGACAGTC BLV1H12-20 CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA SDF1(alpha)GCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCAAGCCCGTCAGCCTGAGCTACAGATGCCCATGCCGATTCTTCGAAAGCCATGTTGCCAGAGCCAACGTCAAGCATCTCAAAATTCTCAACACTCCAAACTGTGCCCTTCAGATTGTAGCCCGGCTGAAGAACAACAACAGACAAGTGTGCATTGACCCGAAGCTAAAGTGGATTCAGGAGTACCTGGAGAAAGCTTTAAACAAGGGCAGCGGTTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGGACAGG GCCTGCTGGTGACAGTC BLV1H12- 21CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA IL21GCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCCAAGGTCAAGATCGCCACATGATCAGAATGCGTCAGCTCATAGATATTGTTGATCAGCTGAAGAACTACGTGAACGACTTGGTCCCTGAATTTCTGCCAGCTCCCGAAGATGTAGAGACAAACTGTGAGTGGTCAGCCTTCTCCTGCTTTCAGAAGGCCCAACTAAAGTCAGCAAATACCGGCAACAACGAGAGGATAATCAATGTATCAATCAAAAAGCTGAAGAGGAAGCCACCTTCCACAAATGCAGGGAGACGGCAGAAACACCGCCTGACATGCCCTTCATGTGATTCTTACGAGAAGAAGCCACCCAAAGAGTTCCTAGAGCGGTTCAAGTCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTCGCACACACGGAAGTGAAGATTCCTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTGC TGGTGACAGTC BLV1H12- 22CAGGTCCAGCTGAGAGAGAGCGGCCCTTCACTGGTCAA ProTxIIGCCATCCCAGACACTGAGCCTGACATGCACAGCAAGCGGGTTTTCACTGAGCGACAAGGCAGTGGGATGGGTCCGACAGGCACCAGGAAAAGCCCTGGAATGGCTGGGCAGCATCGATACCGGCGGGAACACAGGGTACAATCCCGGACTGAAGAGCAGACTGTCCATTACCAAGGACAACTCTAAAAGTCAGGTGTCACTGAGCGTGAGCTCCGTCACCACAGAGGATAGTGCAACTTACTATTGCACCTCTGTGCACCAGGAAACTAAGAAATACCAGAGCTATTGCCAGAAGTGGATGTGGACCTGCGATAGCGAACGGAAATGTTGCGAAGGCATGGTGTGCCGCCTGTGGTGCAAGAAGAAACTCTGGTCTTATACCTACAATTATGAATGGCATGTGGATGTCTGGGGACAGGGCCTG CTGGTGACAGTC

TABLE 21 SEQ ID Name NO Sequence Light 23QAVLNQPSSVSGSLGQRVSITCSGSSSNVGNGYVSWYQLIPGSAP ChainRTLIYGDTSRASGVPDRFSGSRSGNTATLTISSLQAEDEADYFCASAEDSSSNAVFGSGTTLTVLGQPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWKADGSTITRNVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYSCEVTHEGSTVTKTVKPSECS Heavy 24QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG ChainKALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV

IFN- 25 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG betaKALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYC TSVHQETKKYQ S

MSYNLLGFLQRSSNFQC QKLLWQLNGRLEYCLKDRIVINFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTG YLRN

S YTYNYEW HVDVWGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKA

bGCSF- 26 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L0KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYC TSVHQETKKYQ STPLGPARSLPQSFLLKCLEQVRKIQADGAELQERLCAAHKLCHPEELMLLRHSLGIPQAPLSSCSSQSLQLTSCLNQLHGGLFLYQGLLQALAGISPELAPTLDTLQLDVTDFATNIWLQMEDLGAAPAVQPTQGAMPTFTSAFQRRAGGVLVASQLHRFLELAYRG LRYLAEP S YTYNYEWHVDVWGQGLLVTVSSASTTAPKVYPLSS CCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVE

bGCSF- 27 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L1KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYC TSVHQETKKYQ S

TPLGPARSLPQSFLLKCL EQVRKIQADGAELQERLCAAHKLCHPEELMLLRHSLGIPQAPLSSCSSQSLQLTSCLNQLHGGLFLYQGLLQALAGISPELAPTLDTLQLDVTDFATNIWLQMEDLGAAPAVQPTQGAMPTFTSAFQRRAGGVLVASQLHRFL ELAYRGLRYLAEP

S YTYNYEW HVDVWGQGLLVTVSSAS TTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPA

GMCSF 28 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYC TSVHQETKKYQ S

APARSPSPSTQPWEHVN AIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFICENLKDF LLVIPFDCWEPVQE

S YTYNYEW HVDVWGQGLLVTVSSA STTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHP

hFGF21 29 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYC TSVHQETKKYQ S

HPIPDSSPLLQFGGQVR QRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPD VGSSDPLSMVGPSQGRSPSYAS

S YTYNYEW HVDVWGQGL LVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFT

Ex-4 30 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYC TSVHQETKKYQ SC

IEGRHGEGTFTSDLSK QMEEEAVRLFIEWLKNGGPSSGAPPPS

CS YTYNYEW HVDV WGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGS

hGLP-1 31 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV

LEGQAAKEFIAWLVKGR

CS YTYNYEW HVDVWGQGLLVT VSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCN

hEPO 32 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYC TSVHQETKKYQ S

APPRLICDSRVLERYLLE AKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRNIEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACR TGDR

S YTYNYEW HVDVWGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKA

Moka- 33 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L0KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYC TSVHQETKKYQ SINVKCSLPQQCIICPCKDAGMRF GKCMNKKCRCYS S YTYNYEW HVDVWGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKV

Moka- 34 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L1KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYC TSVHQETKKYQ S

INVKCSLPQQCIKPCKDA GMRFGKCMNKKCRCYS

S YTYNYEW HVDVWGQGLLVTV SSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNV

VM-24- 35 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L1KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYC TSVHQETKKYQ S

AAAISCVGSPECPPKCRA QGCKNGKCMNRKCKCYYC

S YTYNYEW HVDVWGQGLLV TVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCN

VM-24- 36 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPG L2KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYC TSVHQETKKYQ S

AAAISCVGSPECPP KCRAQGCKNGKCMNRKCKCYYC

S YTYNYEW HVDV WGQGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGS

Protoxin 37 QVQLRESGPSLKPSQTLSLTCTASGFSLSDKAVGWVRQAPG 2-L1KALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSV TTEDSATYYCTS VHQETKKYQ S

YCQKWIVIWTCDSERKCC EGMVCRLWCKKKLWG

S YTYNYEW HVDVWGQGLLVTVSS ASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAH

For SEQ ID NOS: 24-37 bovine heavy chain sequence = bold human heavychain sequence = dashed underline non-antibody sequence = italic Stalk =bold, underline ; knob = bold, double underline ; linker = italic,squiggly underline

TABLE 22  SEQ ID Name NO SEQUENCE (bold font = non-antibody sequence)BLV1H12-IL8 38 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTSVHQETKKYQSPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRA ENSGSGSYTYNYEWHVDVWGQGLLVTVBLV1H12- 39 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL ZiconotideEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTSVHQETKKYQSCKGKGAKCSRLMYDCCTGSCRSGKCS YTYNYEWHVDVWGQGLLVTV BLV1H12-40 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL SomatostatinEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTSVHQETKKYQSAGCKNFFWKTFTSCGSYTYNYEWHVD VWGQGLLVTV BLV1H12- 41QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL ChlorotoxinEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTSVHQETKKYQSMCMPCFTTDHQMARKCDDCCGGKGR GKCYGPQCLSYTYNYEWHVDVWGQGLLVTVBLV1H12- 42 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL SDF1(alpha)EWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTSVHQETKKYQSKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNKGS GSYTYNYEWHVDVWGQGLLVTVBLV1H12-IL21 43 QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKALEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTSVHQETKKYQSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSYTYNYEWHVDVWGQGLLVTV BLV1H12- 44QVQLRESGPSLVKPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL ProTxIIEWLGSIDTGGNTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTSVHQETKKYQSYCQKWMWTCDSERKCCEGMVCRLW CKKKLWSYTYNYEWHVDVWGQGLLVTVBLV5B8 340 QVQLRESGPSLVQPSQTLSLTCTASGFSLSDKAVGWVRQAPGKAL VHCH1EWLGSIDTGGSTGYNPGLKSRLSITKDNSKSQVSLSVSSVTTEDSATYYCTTVHQETRKTCSDGYIAVDSCGRGQSDGCVNDCNSCYYGWRNCRRQPAIHSYEFHVDAWGRGLLVTVSSASTTAPKVYPLSSCCGDKSSSTVTLGCLVSSYMPEPVTVTWNSGALKSGVHTFPAVLQSSGLYSLSSMVTVPGSTSGQTFTCNVAHPASSTKVDKAVEPKSCDG S BLV5B8 341QAVLNQPSSVSGSLGQRVSITCSGSSSNVGNGYVSWYQLIPGSAP VLCLRTLIYGDTSRASGVPDRFSGSRSGNTATLTISSLQAEDEADYFCASAEDSSSNAVFGSGTTLTVLGQPKSPPSVTLFPPSTEELNGNKATLVCLISDFYPGSVTVVWKADGSTITRNVETTRASKQSNSKYAASSYLSLTSSDWKSKGSYSCEVTHEGSTVTKTVKPSECS

TABLE 23  Exemplary cysteine motifs SEQ ID NO: Sequence 45CX₁₀CX₅CX₅CXCX₇C 46 CX₁₀CX₆CX₅CXCX₁₅C 47 CX₁₁CXCX₅C 48 CX₁₁CX₅CX₅CXCX₇C49 CX₁₀CX₆CX₅CXCX₁₃C 50 CX₁₀CX₅CXCX₄CX₈C 51 CX₁₀CX₆CX₆CXCX₇C 52CX₁₀CX₄CX₇CXCX₈C 53 CX₁₀CX₄CX₇CXCX₇C 54 CX₁₃CX₈CX₈C 55 CX₁₀CX₆CX₅CXCX₇C56 CX₁₀CX₅CX₅C 57 CX₁₀CX₅CX₆CXCX₇C 58 CX₁₀CX₆CX₅CX₇CX₉C 59CX₉CX₇CX₅CXCX₇C 60 CX₁₀CX₆CX₅CXCX₉C 61 CX₁₀CXCX₄CX₅CX₁₁C 62CX₇CX₃CX₆CX₅CXCX₅CX₁₀C 63 CX₁₀CXCX₄CX₅CXCX₂CX₃C 64 CX₁₆CX₅CXC 65CX₆CX₄CXCX₄CX₅C 66 CX₁₁CX₄CX₅CX₆CX₃C 67 CX₈CX₂CX₆CX₅C 68CX₁₀CX₅CX₅CXCX₁₀C 69 CX₁₀CXCX₆CX₄CXC 70 CX₁₀CX₅CX₅CXCX₂C 71CX₁₄CX₂CX₃CXCXC 72 CX₁₅CX₅CXC 73 CX₄CX₆CX₉CX₂CX₁₁C 74 CX₆CX₄CX₅CX₅CX₁₂C75 CX₇CX₃CXCXCX₄CX₅CX₉C 76 CX₁₀CX₆CX₅C 77 CX₇CX₃CX₅CX₅CX₉C 78CX₇CX₅CXCX₂C 79 CX₁₀CXCX₆C 80 CX₁₀CX₃CX₃CX₅CX₇CXCX₆C 81CX₁₀CX₄CX₅CX₁₂CX₂C 82 CX₁₂CX₄CX₅CXCXCX₉CX₃C 83 CX₁₂CX₄CX₅CX₁₂CX₂C 84CX₁₀CX₆CX₅CXCX₁₁C 85 CX₁₆CX₅CXCXCX₁₄C 86 CX₁₀CX₅CXCX₈CX₆C 87CX₁₂CX₄CX₅CX₈CX₂C 88 CX₁₂CX₅CX₅CXCX₈C 89 CX₁₀CX₆CX₅CXCX₄CXCX₉C 90CX₁₁CX₄CX₅CX₈CX₂C 91 CX₁₀CX₆CX₅CX₈CX₂C 92 CX₁₀CX₆CX₅CXCX₈C 93CX₁₀CX₆CX₅CXCX₃CX₈CX₂C 94 CX₁₀CX₆CX₅CXCX₂CX₆CX₅C 95 CX₁₀CX₆CX₅CX₃CX₈C 96CX₇CX₆CX₃CX₃CX₉C 97 CX₉CX₈CX₅CX₆CX₅C 98 CX₁₀CX₂CX₂CX₇CXCX₁₁CX₅C 99CX₁₀CX₆CX₅CXCX₂CX₈CX₄C 100 CCX₃CXCX₃CX₂CCXCX₅CX₉CX₅CXC 101CX₆CX₂CX₅CX₄CCXCX₄CX₆CXC 102 CX₆CX₂CX₅CX₄CCXCX₄CX₆CXC 103CX₉CX₃CXCX₂CXCCCX₆CX₄C 104 CX₅CX₃CXCX₄CX₄CCX₁₀CX₂CC 105CX₅CXCX₁CXCX₃CCX₃CX₄CX₁₀C 106 CX₉CCCX₃CX₄CCCX₅CX₆C 107CCX₈CX₅CX₄CX₃CX₄CCXCX₁C 108 CCX₆CCX₅CCCX₄CX₄CX₁₂C 109CX₆CX₂CX₃CCCX₄CX₅CX₃CX₃C 110 CX₃CX₅CX₆CX₄CCXCX₅CX₄CXC 111CX₄CX₄CCX₄CX₄CXCX₁₁CX₂CXC 112 CX₅CX₂CCX₅CX₄CCX₃CCX₇C 113CX₅CX₅CX₃CX₂CXCCX₄CX₇CXC 114 CX₃CX₇CX₃CX₄CCXCX₂CX₅CX₂C 115CX₉CX₃CXCX₄CCX₅CCCX₆C 116 CX₉CX₃CXCX₂CXCCX₆CX₃CX₃C 117CX₈CCXCX₃CCX₃CXCX₃CX₄C 118 CX₉CCX₄CX₂CXCCXCX₄CX₃C 119CX₁₀CXCX₃CX₂CXCCX₄CX₅CXC 120 CX₉CXCX₃CX₂CXCCX₄CX₅CXC 121CX₆CCXCX₅CX₄CCXCX₅CX₂C 122 CX₆CCXCX₃CXCCX₃CX₄CC 123CX₆CCXCX₃CXCX₂CXCX₄CX₈C 124 CX₄CX₂CCX₃CXCX₄CCX₂CX₃C 125CX₃CX₅CX₃CCCX₄CX₉C 126 CCX₉CX₃CXCCX₃CX₅C 127 CX₉CX₂CX₃CX₄CCX₄CX₅C 128CX₉CX₇CX₄CCXCX₇CX₃C 129 CX₉CX₃CCCX₁₀CX₂CX₃C 130 CX₃CX₅CX₅CX₄CCX₁₀CX₆C131 CX₉CX₅CX₄CCXCX₅CX₄C 132 CX₇CXCX₆CX₄CCCX₁₀C 133 CX₈CX₂CX₄CCX₄CX₃CX₃C134 CX₇CX₅CXCX₄CCX₇CX₄C 135 CX₁₁CX₃CX₄CCCX₈CX₂C 136CX₂CX₃CX₄CCX₄CX₅CX₁₅C 137 CX₉CX₅CX₄CCX₇C 138 CX₉CX₇CX₃CX₂CX₆C 139CX₉CX₅CX₄CCX₁₄C 140 CX₉CX₅CX₄CCX₈C 141 CX₉CX₆CX₄CCXC 142 CX₅CCX₇CX₄CX₁₂143 CX₁₀CX₃CX₄CCX₄C 144 CX₉CX₄CCX₅CX₄C 145 CX₁₀CX₃CX₄CX₇CXC 146CX₇CX₇CX₂CX₂CX₃C 147 CX₉CX₄CX₄CCX₆C 148 CX₇CXCX₃CXCX₆C 149CX₇CXCX₄CXCX₄C 150 CX₉CX₅CX₄C 151 CX₃CX₆CX₈C 152 CX₁₀CXCX₄C 153CX₁₀CCX₄C 154 CX₁₅C 155 CX₁₀C 156 CX₉C

TABLE 24  Exemplary conserved motifs with  the stalk domain SEQ ID NO:Sequence 157 TSVHQETKKYQ 158 VHQETKKYQ 159 TTVHQ 160 TSVHQ 161 SSVTQ 162STVHQ 163 ATVRQ 164 TTVYQ 165 SPVHQ 166 ATVYQ 167 TAVYQ 168 TNVHQ 169ATVHQ 170 STVYQ 171 TIVHQ 172 ATVYQ 173 TTVFQ 174 AAVFQ 175 GTVHQ 176ASVHQ 177 TAVFQ 178 ATVFQ 179 AAAHQ 180 VVVYQ 181 GTVFQ 182 TAVHQ 183ITVHQ 184 ITAHQ 185 VTVHQ 186 AAVHQ 187 GTVYQ 188 TTVLQ 189 TTTHQ 190TTDYQ 191 TTDYQ 192 CTSVHQ 193 CSSVTQ 194 CSTVHQ 195 CATVRQ 196 CTTVYQ197 CSPVHQ 198 CATVYQ 199 CTAVYQ 200 CTNVHQ 201 CATVHQ 202 CSTVYQ 203CTIVHQ 204 CAIVYQ 205 CTTVFQ 206 CAAVFQ 207 CGTVHQ 208 CASVHQ 209 CTAVFQ210 CATVFQ 211 CAAAHQ 212 CVVVYQ 213 CGTVFQ 214 CTAVHQ 215 CITVHQ 216CITAHQ 217 CVTVHQ 218 CAAVHQ 219 CGTVYQ 220 CTTVLQ 221 CTTTHQ 222 CTTDYQ223 CTTVHQX_(n) 224 CTSVHQX_(n) 225 VHQ 226 KKQ 227 VYQ 228 CX¹ X²X³ X⁴Q229 X¹ X²VHQ 230 CX¹ X²VHQ 231 X¹ X²VX³Q 232 CX¹ X²VX³Q 233 X¹ X²KKQ 234CX¹ X²KKQ 235 YTYNYEW 236 YTYNYE 237 YLYTYEH 238 YLYTYE 239 CYTYNYEF 240HYTYTYDF 241 HYTYTYEW 242 KHRYTYEW 243 NYIYKYSF 244 PYIYTYQF 245SFTYTYEW 246 SYIYIYQW 247 SYNYTYSW 248 SYSYSYEY 249 SYTYNYDF 250SYTYNYEW 251 SYTYNYQF 252 SYVWTHNF 253 TYKYVYEW 254 TYTYTYEF 255TYTYTYEW 256 VFTYTYEF 257 AYTYEW 258 DYIYTY 259 IHSYEF 260 SFTYEF 261SHSYEF 262 THTYEF 263 TWTYEF 264 TYNYEW 265 TYSYEF 266 TYSYEH 267 TYTYDF268 TYTYEF 269 TYTYEW 270 AYEF 271 AYSF 272 AYSY 273 CYSF 274 DYTY 275KYEH 276 KYEW 277 MYEF 278 NWIY 279 NYDY 280 NYQW 281 NYSF 282 PYEW 283RYNW 284 RYTY 285 SYEF 286 SYEH 287 SYEW 288 SYKW 289 SYTY 290 TYDF 291TYEF 292 TYEW 293 TYQW 294 TYTY 295 VYEW 296 YX¹YX² 297 YX¹YX² Y 298YX¹YX² YX³ 299 YX¹YX² YX³X⁴ 300 YEX 301 YDX 302 XYE 303 XYD 304YEX¹X_(n)W 305 YDX¹X_(n)W 306 YEX¹X²X³X⁴X⁵W 307 YDX¹X²X³X⁴X⁵W 333 YEXXXW334 YEXXXXW 335 YDXXXW 336 YDXXXXW

TABLE 25  Exemplary Linker Sequences SEQ ID NO: Sequence 337 GGGSGGGGS338 GGGGSGGGS 339 (GGGS)n 342 (GSG)n

TABLE 26  Exemplary non-antibody sequences SEQ ID Description NO:Sequence IL8 317 PRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS ziconotide 318 CKGKGAKCSRLMYDCCTGSCRSGKCsomatostatin 319 AGCKNFFWKTFTSCG chlorotoxin 320MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCL SDF1(alpha) 321KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLK NNNRQVCIDPKLKWIQEYLEKALNKIL21 322 QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGS EDS Protoxin2 323YCQKWMWTCDSERKCCEGMVCRLWCKKKLW IFN-beta 324MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN bGCSF 325TPLGPARSLPQSFLLKCLEQVRKIQADGAELQERLCAAHKLCHPEELMLLRHSLGIPQAPLSSCSSQSLQLTSCLNQLHGGLFLYQGLLQALAGISPELAPTLDTLQLDVTDFATNIWLQMEDLGAAPAVQPTQGAMPTFTSAFQRRAGGVLVASQLHRFLELAYRGLRYLA EP GMCSF 326APARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE hFGF21 327HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPAPPEPPGILAPQPPDVGSSDPLSMVGPSQGR SPSYAS Ex-4 328HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS hGLP-1 329HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR hEPO 330PPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR Moka 331INVKCSLPQQCIKPCKDAGMRFGKCMNKKCRCYS VM-24 332AAAISCVGSPECPPKCRAQGCKNGKCMNRKCKCYYC

1. A library of antibodies or binding fragments thereof, wherein theantibodies or binding fragments thereof comprise an ultralong CDR3.
 2. Alibrary of polynucleotides encoding for antibodies or binding fragmentsthereof, wherein the encoded antibodies or binding fragments thereofcomprise an ultralong CDR3. 3-47. (canceled)
 48. A recombinant antibodyor fragment thereof, wherein at least a portion of the recombinantantibody or fragment thereof is based on or derived from at least aportion of an ultralong CDR3.
 49. An antibody or fragment thereofcomprising: (a) a first antibody sequence, wherein at least a portion ofthe first antibody sequence is derived from at least a portion of anultralong CDR3; (b) a non-antibody sequence; and (c) optionally, asecond antibody sequence, wherein at least a portion of the secondantibody sequence is derived from at least a portion of an ultralongCDR3. 50-158. (canceled)
 159. A library of antibodies or bindingfragments thereof, wherein the antibodies or binding fragments thereofcomprise an ultralong CDR3.
 160. A library of antibodies or bindingfragments thereof, wherein the antibodies or binding fragments thereofcomprise the antibody or binding fragment of claim
 48. 161. A nucleicacid library comprising a plurality of polynucleotides comprisingsequences coding for antibodies or binding fragments thereof, whereinthe antibodies or binding fragments thereof comprise an ultralong CDR3.162. A nucleic acid library comprising a plurality of polynucleotidescomprising sequences coding for antibodies or binding fragments thereof,wherein the antibodies or binding fragments thereof comprise theantibody or binding fragment of claim
 48. 163-165. (canceled)
 166. Apolynucleotide comprising a nucleic acid sequence that encodes theantibody or binding fragment thereof of claim
 48. 167. A vectorcomprising a polynucleotide, wherein the polynucleotide comprises anucleic acid sequence that encodes the antibody or binding fragmentthereof of claim
 48. 168. A host cell comprising a polynucleotide,wherein the polynucleotide comprises a nucleic acid sequence thatencodes the antibody or binding fragment thereof of claim
 48. 169. Amethod of producing an antibody or binding fragment thereof comprisingan ultralong CDR3 or fragment thereof comprising culturing the host cellof claim 168 under conditions wherein the polynucleotide sequence isexpressed and the antibody or binding fragment thereof comprising anultralong CDR3 or fragment thereof is produced.
 170. (canceled)
 171. Apharmaceutical composition comprising an antibody or fragment thereof ofclaim
 48. 172. A pharmaceutical composition comprising (a) an antibodyor fragment thereof comprising sequence based on or derived from atleast a portion of an ultralong CDR3; and (b) a pharmaceuticallyacceptable excipient. 173-174. (canceled)
 175. A method of treating adisease or condition in a subject in need thereof comprisingadministering to the mammal a therapeutically effective amount of theantibody of claim
 48. 176-188. (canceled)
 189. A library of antibodiesor binding fragments thereof, wherein the antibodies or bindingfragments thereof comprise the antibody or binding fragment of claim 49.190. A nucleic acid library comprising a plurality of polynucleotidescomprising sequences coding for antibodies or binding fragments thereof,wherein the antibodies or binding fragments thereof comprise theantibody or binding fragment of claim
 49. 191. A polynucleotidecomprising a nucleic acid sequence that encodes the antibody or bindingfragment thereof of claim
 49. 192. A vector comprising a polynucleotide,wherein the polynucleotide comprises a nucleic acid sequence thatencodes the antibody or binding fragment thereof of claim
 49. 193. Ahost cell comprising a polynucleotide, wherein the polynucleotidecomprises a nucleic acid sequence that encodes the antibody or bindingfragment thereof claim
 49. 194. A method of producing an antibody orbinding fragment thereof comprising an ultralong CDR3 or fragmentthereof comprising culturing the host cell of claim 193 under conditionswherein the polynucleotide sequence is expressed and the antibody orbinding fragment thereof comprising an ultralong CDR3 or fragmentthereof is produced.